Methods of Diagnosing Intervertebral Disc Disease And Chondrodystrophy In Canines

ABSTRACT

Provided are compositions and methods for identifying a canine suffering from or at risk of suffering from skeletal dysplasia (SD) and/or intervertebral disc disease (IVDD) by detecting a retrogene insertion encoding canine fibroblast growth factor 4 (FGF4) on canine chromosome 12.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/512,689, filed on May 30, 2017, which is hereby incorporated hereinby reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Grant No. NIH 5T32 OD010931 2016_20F, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Variation in domestic dog (Canis familiaris, CFA) morphology has longfascinated both scientists and pet owners. Domestication of the dog fromthe wolf and the subsequent variation in size and shape within purebreddog breeds is a remarkable feat of animal breeding and selection. One ofthe most extreme examples of dog breed differences is in limb length, asextremely short limbs define many breeds. This morphological feature ispresent in breeds from all over the world and from all American KennelClub groups, indicating that the underlying genetic causes are likelyvery old.

Extensive examination of growth plates has been performed on many ofthese short-legged dog breeds (Dachshund, Pekingese, French Bulldog,Spaniels, Beagle), as these breeds are also prone to intervertebral discdisease (IVDD) (1-3). Histopathological analysis of the bones of puppiesfrom these breeds demonstrated that their short stature is due todefects in endochondral ossification, the process whereby cartilage isreplaced with bone in the developing limb. The long bone growth platesshow disorganization of the proliferative zone and reduction in thedepth of the maturation zone (1-4). In addition to the long bones,similar but more subtle changes exist in endochondral ossification ofthe vertebral bodies (1,2).

The intervertebral disc (IVD) is composed of an outer fibrous basket,called the annulus fibrosis, made of 70% collagen and an inner gel-likelayer that is a remnant of the embryonic notochord, called the nucleuspulposus (5). Together, these structures and the cartilaginous endplatesallow for flexibility of the vertebral column. In chondrodystrophicdogs, the nucleus pulposus is gradually replaced by chondrocyte-likecells in chondroid metaplasia (or metamorphosis) that occurs betweenbirth and 1 year of age (1,2). Recent studies have shown that inadvanced stages of degeneration in non-chondrodystrophoid dogs there isalso replacement of notochordal cells by chondrocyte-like cells, similarto the changes observed in chondrodystrophoid dogs, although thishappens at an older age (3,6-10). The replacement of the nucleuspulposus with chondrocyte like cells is seen in humans, andchondrodystrophoid breeds have been proposed as models for humandegenerative disc disease (3, 7, 11, 12).

Hansen described the two different types of canine IVD prolapse as typeI and type II. Type I occurs exclusively in chondrodystrophic breeds andis characterized by premature degeneration of all discs in young dogs.In contrast, Type II occurs in older dogs and is usually limited to asingle disc with only partial protrusion. In Type I disc disease thecalcified nucleus pulposus may undergo an explosive herniation throughthe annulus fibrous into the vertebral canal, resulting in inflammationand hemorrhage and causing severe pain and neurological dysfunction (1,2). In veterinary hospital population studies, breeds with a significantincreased risk of IVDD include the Beagle, Cocker Spaniel, Dachshund,French Bulldog, Lhasa Apso, Pekingese, Pembroke Welsh Corgi, and ShihTzu (13-15). Pet insurance data suggests a conservative “lifetimeprevalence” for IVDD in dogs of 3.5% in the overall population; however,in the chondrodystrophic breed with the highest risk, the Dachshund, the“lifetime prevalence” is between 20-62% with a mortality rate of 24% (9,16-19). The effect of this disease on dogs and the financial burden topet owners is enormous.

Skeletal dysplasia (SD), a general term to classify abnormalities ofgrowth and development of cartilage and/or bone resulting in variousforms of short stature, occurs in humans and dogs in many forms (20).With advances in molecular genetics, many of the diseases in humans arebeing reclassified based on the specific underlying causative mutations(21). To a lesser degree, progress has also been made in understandingthe molecular nature of SD and the extreme interbreed limb lengthvariation observed in dogs (22-25). While the mutations causing somesubtypes of SD in dogs have been determined, there are still manyunexplained types of SD observed within and across dog breeds.

In 2009 the genetic basis for extreme differences in limb length in dogswas investigated by Parker et al. using an across breed genome-wideassociation approach (26). They determined that a FGF4 retrogeneinsertion on CFA18 was responsible for the “chondrodysplasia” phenotypein a number of breeds, such as the Basset Hound, Pembroke Welsh Corgi,and Dachshund. However, the FGF4 retrogene insertion on CFA18 failed toexplain breeds such as the American Cocker Spaniel, Beagle, and FrenchBulldog, that in addition to Dachshunds, were the breeds originallyclassified as chondrodystrophoid based on histopathologic andmorphologic analysis by Hansen and Braund (1, 3). The FGF gene familyhas similarly been implicated in SD in humans, with mutations in FGFR3found to be responsible for achondrodysplasia, the most common form ofdwarfism, characterized by shortened limbs and abnormal vertebrae andIVDs (21, 27-31). FGF genes are involved in a number of embryologicaldevelopment processes, and specific levels of ligand and receptor arekey for appropriate growth and development (32-34).

SUMMARY

In one aspect, provided are reaction mixtures. In some embodiments, thereaction mixtures comprise (i) a biological sample from a caninecomprising a nucleic acid template, and (ii) one or more oligonucleotidepairs configured to detect the presence or absence of a retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) on caninechromosome 12. In some embodiments, the retrogene comprises about 3.2kilobases (kb). In some embodiments, retrogene insertion is inserted ata target site duplication sequence located atchr12:33,710,168-33,710,178 (canFam3). In some embodiments, theoligonucleotide pairs detect the 5′-end and/or the 3′-end of theretrogene insertion. In some embodiments, the 5′-end of the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) comprises anucleic acid sequence having at least 90% sequence identity, e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to nucleic acid residues 1002-2001 SEQ ID NO:1. In someembodiments, the one or more oligonucleotide pairs are configured ordesigned or constructed to detect the 5′-end of the retrogene insertionlocated at nucleic acid residue 1002 of SEQ ID NO:1. In someembodiments, an oligonucleotide in the one or more oligonucleotide pairshybridizes to a sequence segment within nucleic acid residues 1-1001 ofSEQ ID NO:1. In some embodiments, the 3′-end of the retrogene insertionencoding canine fibroblast growth factor 4 (FGF4) comprises a nucleicacid sequence having at least 90% sequence identity, e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, tonucleic acid residues 1-1000 SEQ ID NO:2. In some embodiments, the oneor more oligonucleotide pairs are configured to detect the 3′-end of theretrogene insertion located at nucleic acid residue 1000 of SEQ ID NO:2.In some embodiments, an oligonucleotide in the one or moreoligonucleotide pairs hybridizes to a sequence segment within nucleicacid residues 1001-2000 of SEQ ID NO:2. In some embodiments, the one ormore oligonucleotide pairs comprise one or more forward primers selectedfrom the group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2),GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4),AGCCTGATGGCTGGACTGTA (SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQ IDNO:6) and one or more reverse primers selected from the group consistingof TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7), CCTGATTTTGAGACAGCCAAA (SEQ IDNO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9) and TGAGTGGGTTAAGGGTTTCG (SEQID NO:10). In some embodiments, the one or more oligonucleotidescomprise one or more forward primers selected from the group consistingof:

(SEQ ID NO: 2) ACAGCTGGCATGGTCAGTTA and (SEQ ID NO: 6)GTCCGTGCGGTGAAATAAAA and reverse primer (SEQ ID NO: 7)TGCTGTAGATTTTGAGGTGTCTT.In some embodiments, the nucleic acid template comprises genomic DNA. Insome embodiments, the reaction mixture further comprises a polymeraseand dNTPs.

In another aspect, provided are kits. In some embodiments, the kitscomprise one or more oligonucleotide pairs that specifically identifythe presence or absence of a retrogene insertion encoding caninefibroblast growth factor 4 (FGF4) on canine chromosome 12. In someembodiments, the oligonucleotide pairs detect the 5′-end and/or the3′-end of the retrogene insertion. In some embodiments, the 5′-end ofthe retrogene insertion encoding canine fibroblast growth factor 4(FGF4) comprises a nucleic acid sequence having at least 90% sequenceidentity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, to nucleic acid residues 1002-2001 SEQ ID NO:1.In some embodiments, the one or more oligonucleotide pairs areconfigured to detect the 5′-end of the retrogene insertion located atnucleic acid residue 1002 of SEQ ID NO:1. In some embodiments, anoligonucleotide in the one or more oligonucleotide pairs hybridizes to asequence segment within nucleic acid residues 1-1001 of SEQ ID NO:1. Insome embodiments, the 3′-end of the retrogene insertion encoding caninefibroblast growth factor 4 (FGF4) comprises a nucleic acid sequencehaving at least 90% sequence identity, e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to nucleic acidresidues 1-1000 SEQ ID NO:2. In some embodiments, the one or moreoligonucleotide pairs are configured to detect the 3′-end of theretrogene insertion located at nucleic acid residue 1000 of SEQ ID NO:2.In some embodiments, an oligonucleotide in the one or moreoligonucleotide pairs hybridizes to a sequence segment within nucleicacid residues 1001-2000 of SEQ ID NO:2. In some embodiments, the one ormore oligonucleotide pairs comprise one or more forward primers selectedfrom the group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2),GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4),AGCCTGATGGCTGGACTGTA (SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQ IDNO:6) and one or more reverse primers selected from the group consistingof TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7), CCTGATTTTGAGACAGCCAAA (SEQ IDNO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9) and TGAGTGGGTTAAGGGTTTCG (SEQID NO:10).

In another aspect, provided are solid supports. In some embodiments, thesolid supports are attached to one or more oligonucleotides thatspecifically identify the presence or absence of a retrogene insertionencoding canine fibroblast growth factor 4 (FGF4) on canine chromosome12. In some embodiments, the solid support is attached to anoligonucleotide that hybridizes to the 5′-end of the retrogene insertionlocated at nucleic acid residue 1002 of SEQ ID NO:1. In someembodiments, the solid support is attached to an oligonucleotide thathybridizes to the 3′-end of the retrogene insertion located at nucleicacid residue 1000 of SEQ ID NO:2. In some embodiments, the solid supportis attached to an oligonucleotide having at least about 80% sequenceidentity, e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to SEQ ID NO:11 and/or SEQ ID NO:12. In some embodiments, thesolid support is a microarray. In some embodiments, the solid support isa mounted tissue sample. Further provided are kits comprising the solidsupport as described above and herein.

In a further aspect, provided are methods for identifying a caninesuffering from or at risk of suffering from skeletal dysplasia (SD)and/or intervertebral disc disease (IVDD). In some embodiment, themethods comprise: a) obtaining a biological sample comprising a nucleicacid template from the canine; b) determining the presence or absence ofa retrogene insertion encoding canine fibroblast growth factor 4 (FGF4)on canine chromosome 12; and c) selecting a canine comprising theretrogene insertion identifies a canine suffering from or at risk ofsuffering from skeletal dysplasia (SD) and/or intervertebral discdisease (IVDD) relative to canine that does not have the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) on caninechromosome 12. In a related aspect, provided are methods for identifyinga canine with reduced risk of suffering from skeletal dysplasia (SD)and/or intervertebral disc disease (IVDD). In some embodiments, themethods comprise: a) obtaining a biological sample comprising a nucleicacid template from the canine; b) determining the presence or absence ofa retrogene insertion encoding canine fibroblast growth factor 4 (FGF4)on canine chromosome 12; and c) selecting a canine that does notcomprise the retrogene insertion identifies a canine with reduced riskof suffering from skeletal dysplasia (SD) and/or intervertebral discdisease (IVDD) relative to canine that has the retrogene insertionencoding canine fibroblast growth factor 4 (FGF4) on canine chromosome12. In some embodiments, the retrogene comprises about 3.2 kilobases(kb). In some embodiments, the retrogene insertion is inserted at atarget site duplication sequence located at chr12:33,710,168-33,710,178(canFam3). In some embodiments, the determining step employs one or morepolynucleotides configured to detect the 5′-end and/or the 3′-end of theretrogene insertion. In some embodiments, the 5′-end of the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) comprises anucleic acid sequence having at least 90% sequence identity, e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to nucleic acid residues 1002-2001 SEQ ID NO:1. In someembodiments, the one or more polynucleotides are configured to detectthe 5′-end of the retrogene insertion located at nucleic acid residue1002 of SEQ ID NO:1. In some embodiments, one polynucleotide hybridizesto a sequence segment within nucleic acid residues 1-1001 of SEQ IDNO:1. In some embodiments, the 3′-end of the retrogene insertionencoding canine fibroblast growth factor 4 (FGF4) comprises a nucleicacid sequence having at least 90% sequence identity, e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, tonucleic acid residues 1-1000 SEQ ID NO:2. In some embodiments, the oneor more polynucleotides are configured to detect the 3′-end of theretrogene insertion located at nucleic acid residue 1000 of SEQ ID NO:2.In some embodiments, one polynucleotide hybridizes to a sequence segmentwithin nucleic acid residues 1001-2000 of SEQ ID NO:2. In someembodiments, the SD/IVDD genotype is detected by an amplificationreaction using polynucleotides that identify the presence or absence ofthe CFA 12 FGF4 retrogene insertion. In some embodiments, theamplification reaction is selected from the group consisting ofpolymerase chain reaction (PCR), strand displacement amplification(SDA), nucleic acid sequence based amplification (NASBA), rolling circleamplification (RCA), T7 polymerase mediated amplification, T3 polymerasemediated amplification and SP6 polymerase mediated amplification. Insome embodiments, a portion of the retrogene insertion sequence isspecifically amplified using one or more forward primers selected fromthe group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2),GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4),AGCCTGATGGCTGGACTGTA (SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQ IDNO:6) and one or more reverse primers selected from the group consistingof TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7), CCTGATTTTGAGACAGCCAAA (SEQ IDNO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9) and TGAGTGGGTTAAGGGTTTCG (SEQID NO:10). In some embodiments, a portion of the retrogene insertionsequence is specifically amplified using one or more forward primersselected from the group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ IDNO:2) and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and reverse primerTGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7). In some embodiments, the SD/IVDDgenotype is detected by hybridization using polynucleotides whichidentify the presence or absence of the CFA 12 FGF4 retrogene insertion.In some embodiments, the SD/IVDD genotype is detected by sequencing. Insome embodiments, the canine is a domesticated canine. In someembodiments, the canine is of a breed having a predisposition tochondrodystrophy. In some embodiments, the canine is a purebred or mixfrom a breed selected from the group consisting of American CockerSpaniel, Basset Hound, Beagle, Cardigan Welsh Corgi, Chesapeake BayRetriever, Chihuahua, Coton de Tulear, Dachshund, English SpringerSpaniel, French Bulldog, Jack Russell Terrier, Miniature Schnauzer, NovaScotia Duck Tolling Retriever, Pekingese, Pembroke Welsh Corgi, Poodle,Portuguese Water Dog, Scottish Terrier, Shih Tzu, and mixtures thereof.In varying, the canine is a purebred or mix from a breed selected fromthe group consisting of American Cocker Spaniel, Basset Hound, Beagle,Corgi, Dachshund, French bulldog, Nova Scotia Duck Tolling Retriever,and Pekingese.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art. Generally, the nomenclature used herein andthe laboratory procedures in cell culture, molecular genetics, organicchemistry and nucleic acid chemistry and hybridization described beloware those well-known and commonly employed in the art. Standardtechniques are used for nucleic acid and peptide synthesis. Generally,enzymatic reactions and purification steps are performed according tothe manufacturer's specifications. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (see generally, Green and Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed. (2012) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel, ed.,Current Protocols in Molecular Biology, 1990-2017, John WileyInterscience), which are provided throughout this document. Thenomenclature used herein and the laboratory procedures in analyticalchemistry, and organic synthetic described below are those well-knownand commonly employed in the art. Standard techniques, or modificationsthereof, are used for chemical syntheses and chemical analyses.

“Chondrodystrophy” refers to defects in long bone and vertebral bodyendochondral ossification and abnormal intervertebral discs thatprematurely degenerate and calcify, which ultimately can lead to discherniation and paralysis referred to as Hansen's Type I intervertebraldisc disease (IVDD). Reviewed in, e.g., Smith, et al., Vet Comp OrthopTraumatol. (2016) 29(3):220-6; Smolders, et al., Vet J. (2013)195(3):292-9; Bergknut, et al., Vet J. (2013) 195(3):282-91; Bergknut,et al., Vet J. (2013) 195(2):156-63 and Beachley, et al., J Am Vet MedAssoc. (1973) 163(3):283-4.

“Chondrodysplasia” refers to disproportionate dwarfism.

As used herein, the terms “dog,” “canine” and “Canis lupus familiaris”are used interchangeably.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A “variant” is a difference in the nucleotide sequence among relatedpolynucleotides. The difference may be the deletion of one or morenucleotides from the sequence of one polynucleotide compared to thesequence of a related polynucleotide, the addition of one or morenucleotides or the substitution of one nucleotide for another. The terms“mutation,” “polymorphism” and “variant” are used interchangeably hereinto describe such variants. As used herein, the term “variant” in thesingular is to be construed to include multiple variances; i.e., two ormore nucleotide additions, deletions and/or substitutions in the samepolynucleotide. A “point mutation” refers to a single substitution ofone nucleotide for another.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point I for the specific sequence at a definedionic strength pH. The Tm is the temperature (under defined ionicstrength, pH, and nucleic concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, optionally 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. The term“purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., share at least about 80% identity, for example, at least about85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region to a reference sequence, e.g., SEQ ID NOs:1-11 and theother nucleic acid sequences provided herein), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Such sequencesare then said to be “substantially identical.” This definition alsorefers to the compliment of a test sequence. Preferably, the identityexists over a region that is at least about 25 amino acids ornucleotides in length, for example, over a region that is 50-100 aminoacids or nucleotides in length, or over the full-length of a referencesequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. For sequence comparison of nucleicacids and proteins to canine FGF4 nucleic acids and proteins, the BLASTand BLAST 2.0 algorithms and the default parameters discussed below areused.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

“Array” as used herein refers to a solid support comprising attachednucleic acid or peptide probes. Arrays typically comprise a plurality ofdifferent nucleic acid or peptide probes that are coupled to a surfaceof a substrate in different, known locations. These arrays, alsodescribed as “microarrays” or colloquially “chips” have been generallydescribed in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934,5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., Science,251:767-777 (1991). These arrays may generally be produced usingmechanical synthesis methods or light directed synthesis methods whichincorporate a combination of photolithographic methods and solid phasesynthesis methods. Techniques for the synthesis of these arrays usingmechanical synthesis methods are described in, e.g., U.S. Pat. No.5,384,261. Arrays may comprise a planar surface or may be nucleic acidsor peptides on beads, gels, polymeric surfaces, fibers such as fiberoptics, glass or any other appropriate substrate as described in, e.g.,U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992.Arrays may be packaged in such a manner as to allow for diagnostics orother manipulation of an all-inclusive device, as described in, e.g.,U.S. Pat. Nos. 5,856,174 and 5,922,591.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C illustrate Skeletal Dysplasia (SD) in the Nova Scotia DuckTolling Retriever (NSDTR): a) Picture and lateral thoracic limbradiograph of unaffected NSDTRs (ages 1 year old and 4 years old,respectively). b) Left panels depict picture and lateral thoracic limbradiograph of mildly SD affected NSDTRs (ages 4 years old and 2 yearsold, respectively); right panels depict picture and lateral thoraciclimb radiograph of more severely SD affected NSDTRs (ages 3 years oldand 6 months old, respectively). Relative to the unaffected dog, themildly SD affected NSDTR has cranial bowing of the radius. Radiographicchanges in the more severely SD NSDTR include moderate cranial bowing ofthe radius, physeal widening, and incongruity of the elbow joint withthe shape of the semi-lunar notch of the ulna being elongated. Picturesand radiographs are representatives of each phenotype and not paired(i.e. the radiographs are not of the dogs pictured). c) SD in NSDTRGWAS: Manhattan plot showing log 10 of the raw p-values for eachgenotyped SNP by chromosome (x-axis). After SNP quality control, therewere 106,303 SNPs for Chi square analysis. Genomic inflation was1.01604. Line denotes genome-wide significance based on Bonferronicorrected p-values.

FIG. 2 illustrates quantile-quantile (QQ) plot shows −log 10 of theexpected versus observed p-values plotted for each SNP, with the SNPs onCFA12 colored in grey.

FIGS. 3A-C illustrate across breed investigation of SD-IVDD locus: a)Minor allele frequency on the y-axis and base pair on CFA12 on thex-axis plotted by breed: SD affected NSDTR (n=13), American CockerSpaniel (n=7), and Beagle (n=14). b) Manhattan plot for the SNPs in theacross breed IVDD GWAS showing −log 10 of the raw p-values (y-axis) foreach genotyped SNP by chromosome (x-axis). After SNP quality control,there were 126,020 SNPs for Chi square analysis. Genomic inflation was1.6339. c) SNPs in 5 Mb region surrounding most highly associated SNP(chr12:36,909,311 (canFam2)) plotted by base pair on the x-axis andp-value on the y-axis. SNPs have been pruned from analysis usingrecommended criteria by Kierczak et al. (37). SNPs are color-coded by r²value to show the extent of linkage disequilibrium.

FIG. 4 illustrates large insertion identified on CFA12: Screenshot ofthe Integrative Genomics Viewer (IGV-Broad Institute) showing aninsertion at approximately 12:33,710,200 (canFam3) in an IVDD affectedDachshund case and a SD affected NSDTR that is not present in the Salukiunaffected control. Read mates in green map to chr18:48.4 Mb (canFam3)and the read mates in blue map to chr7:68.3 Mb (canFam3).

FIG. 5 illustrates a schematic of endogenous FGF4 (CFA18)retrotransposition to CFA12:33,710,178 (canFam3): Predicted TATA box atchr12:33,709,940-947 (canFam3) and predicted RNA Pol II promoter atchr12:33,709,964-976 (canFam3), which are 239 bp and 215 bp upstream,respectively (38). Endogenous FGF4 untranslated regions (UTR) areunknown in the dog; however, they were approximated in the figure basedon human and mouse TransMap data available at the UCSC genome browser(genome.ucsc.edu). The predicted 5′UTR spans fromchr18:48,413,185-48,413,480 (canFam3); however, RT-PCR in IVD from aBeagle suggest that the TSS is located betweenchr18:48,413,315-48,413,402 (canFam3). The insert includes all predicted3′UTR, followed by 42 adenine residues and the duplicated 11 bp TSDsequence (AAG TGC TTT GA) (chr12:33,710,168-33,710,178 (canFam3)).Endogenous FGF4 sequence that was retrotransposed also includes a largeCpG island.

FIGS. 6A-C illustrate association of FGF4 insertion genotypes withheight and IVDD: a) Genotyping results for CFA18 and CFA12 FGF4insertions across breeds. Arrows indicate wild type (WT) band. Laneorder: Ladder; 1-3) NSDTR; 4) Beagle; 5) American Cocker Spaniel; 6)Dachshund; 7) Basset Hound; 8) Pembroke Welsh Corgi; 9) Coton de Tulear;10) Cairn Terrier; 11) West Highland White Terrier. b) Height at thewithers in inches (in) was available for 7 SD NSDTR cases: all werehomozygous mutant for the CFA12 FGF4 insertion, and their mean heightwas 18.22 in. Height was available for 13 NSDTR unaffected with SD: 5were wild type and had a mean height of 20.2 in; 8 were heterozygous forthe CFA12 FGF4 insertion and had a mean height of 18.94 in. * indicaterelative levels of association of the insertion with height. ***:p=0.007, **: p=0.016, *: p=0.034. c) Association of various identifiedloci with IVDD, including Chi square, p-value, and Odds ratio (95%confidence intervals in parenthesis).

FIG. 7 illustrates CFA 12 FGF4 Genotypes Across Breeds: Genotypes forthe CFA12 FGF4 insertion across dog breeds ordered by breed standardheight from shortest to tallest (x-axis), plotted by dog weight inkilograms (kg) (y-axis). Only genotyped dogs with weights available(n=376) were included in the figure. Dogs are color-coded by genotypestatus.

FIG. 8 illustrates semi-quantitative RT-PCR for FGF4 across tissues in acase and control. Lane order: Ladder; 1) Control VB (Cane Corso); 2)Case VB (Beagle); 3) Control IVD (Cane Corso); 4) Case IVD (Beagle); 5)Control skeletal muscle (Labrador retriever); 6) Case skeletal muscle(Beagle); 7) Control testis (Labrador retriever); 8) Case testis(Beagle); 9) Negative control.

FIG. 9 illustrates FGF4 expression: Bar graph depicting fold changedifferences in FGF4 expression between controls and IVDD cases inneonatal IVD and VB. FGF4 expression was 19.47× higher (p=0.02857) inIVD and 2.16× higher (p=0.02857) in VB of cases compared to controls.Error bars representative of standard error of measurement for eachgroup. Gels depict genotypes of 4 cases (Beagles) and 5 controls (1Rottweiler and 4 Cane Corso) used in qRT-PCR analysis. The five controlswere wild type, meaning they lacked the FGF4 insert at both the CFA12and CFA18 locations; however, the cases, while wild type for the CFA18FGF4 insert, were homozygous mutant at the CFA12 locus. Lanes: 1-4:Beagle cases; 5-9: Cane Corso controls; 10: heterozygous control; 11:negative control.

DETAILED DESCRIPTION 1. Introduction

Chondrodystrophy in dogs is defined by dysplastic, shortened long bonesand premature degeneration and calcification of intervertebral discs.Independent genome-wide association analyses for skeletal dysplasia(short limbs) within a single breed (pBonferroni=0.0072) andintervertebral disc disease (IVDD) across breeds)(pBonferroni=4.02×10⁻¹⁰ both identified a significant association to thesame region on CFA12. Whole genome sequencing identified a highlyexpressed FGF4 retrogene within this shared region. The FGF4 retrogenesegregated with limb length and had an odds ratio of 51.23 (95%CI=46.69, 56.20) for IVDD. Long bone length in dogs is a unique exampleof multiple disease-causing retrocopies of the same parental gene in amammalian species. FGF signaling abnormalities have been associated withskeletal dysplasia in humans, and our findings present opportunities forboth selective elimination of a medically and financially devastatingdisease in dogs and further understanding of the ever-growing complexityof retrogene biology.

In this study, genome-wide association analysis in a cohort of NovaScotia Duck Tolling Retrievers (NSDTRs) with and without severe SDidentified a significant association on CFA12 due to a 12 Mb associatedhaplotype, of which 1.9 Mb was found to be shared in chondrodystrophoidbreeds. Subsequent genome-wide association analysis of Hansen's type IIVDD across breeds localized the same 1.9 Mb region on CFA12, suggestingthat the locus responsible for SD in the NSDTR is also responsible fortype I IVDD and the chondrodystrophoid phenotype across dog breeds. Aprevious genetic investigation of IVDD in Dachshunds and limb lengthmorphology in Portuguese Water Dogs both identified the same CFA12locus; however, neither study reported a causative mutation (35,36). Thepresent compositions and methods are based, in part, on the discovery ofa second FGF4 retrogene insertion (chr12:33.7 Mb (canFam3)) in thecanine genome and show that it is not only responsible for SD in theNSDTR, but also chondrodystrophy, including the predisposition toHansen's type I IVDD, across all dog breeds.

2. Reaction Mixtures

Provided are reaction mixtures for identifying the presence or absenceof a retrogene insertion encoding canine fibroblast growth factor 4(FGF4) on canine chromosome 12, as correlated with canine skeletaldysplasia (SD) and/or intervertebral disc disease (IVDD). In someembodiments, the reaction mixtures comprise (i) a biological sample froman canine comprising a nucleic acid template, and (ii) one or moreoligonucleotide pairs configured to detect the presence or absence of aretrogene insertion encoding canine fibroblast growth factor 4 (FGF4) oncanine chromosome 12. In some embodiments, the retrogene comprises about3.2 kilobases (kb). In some embodiments, retrogene insertion is insertedat a target site duplication sequence located atchr12:33,710,168-33,710,178 (canFam3). In some embodiments, theoligonucleotide pairs detect the 5′-end and/or the 3′-end of theretrogene insertion. In some embodiments, the 3′-end of the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) comprises anucleic acid sequence having at least 90% sequence identity, e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to nucleic acid residues 1002-2001 SEQ ID NO:1. In someembodiments, the one or more oligonucleotide pairs are configured ordesigned or constructed to detect the 3′-end of the retrogene insertionlocated at nucleic acid residue 1002 of SEQ ID NO:1. In someembodiments, the 5′-end of the retrogene insertion encoding caninefibroblast growth factor 4 (FGF4) comprises a nucleic acid sequencehaving at least 90% sequence identity, e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to nucleic acidresidues 1-1000 SEQ ID NO:2. In some embodiments, the one or moreoligonucleotide pairs are configured to detect the 5′-end of theretrogene insertion located at nucleic acid residue 1000 of SEQ ID NO:2.In some embodiments, the one or more oligonucleotide pairs comprise oneor more forward primers selected from the group consisting of:ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2), GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3),CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4), AGCCTGATGGCTGGACTGTA (SEQ ID NO:5)and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and one or more reverse primersselected from the group consisting of TGCTGTAGATTTTGAGGTGTCTT (SEQ IDNO:7), CCTGATTTTGAGACAGCCAAA (SEQ ID NO:8), TTGATGCCCAGGAGGTAGTC (SEQ IDNO:9) and TGAGTGGGTTAAGGGTTTCG (SEQ ID NO:10). In some embodiments, theone or more oligonucleotides comprise one or more forward primersselected from the group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ IDNO:2) and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and reverse primerTGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7). In some embodiments, the nucleicacid template comprises genomic DNA.

The nucleic acid template in the biological sample can comprise genomicDNA. In some embodiments, the reaction mixtures further can compriseappropriate buffers, salts, polymerases, reverse-transcriptases, dNTPs,nuclease inhibitors, and other reagents to facilitate amplificationand/or detection reactions (e.g., primers, labels) for amplifying thecanine FGF4 retrogene from genomic DNA.

3. Solid Supports

Further provided are solid supports attached to one or morepolynucleotides or oligonucleotides that specifically detect thepresence or absence of a retrogene insertion encoding canine fibroblastgrowth factor 4 (FGF4) on canine chromosome 12, found to correlate withcanine skeletal dysplasia (SD) and/or intervertebral disc disease(IVDD).

In some embodiments, the solid supports are attached to one or moreoligonucleotides that specifically identify the presence or absence of aretrogene insertion encoding canine fibroblast growth factor 4 (FGF4) oncanine chromosome 12. In some embodiments, the solid support is attachedto an oligonucleotide that hybridizes to the 5′-end of the retrogeneinsertion located at nucleic acid residue 1002 of SEQ ID NO: 1. In someembodiments, the solid support is attached to an oligonucleotide thathybridizes to the 3′-end of the retrogene insertion located at nucleicacid residue 1000 of SEQ ID NO:2. In some embodiments, the solid supportis attached to an oligonucleotide having at least about 80% sequenceidentity, e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to SEQ ID NO:11 and/or SEQ ID NO:12. In some embodiments, thesolid support is a microarray. In some embodiments, the solid support isa mounted tissue sample.

In certain embodiments, the solid support is a microarray, e.g., agenotyping array. Microarrays suitable for genotyping are commerciallyavailable, e.g., Axiom™ Canine Genotyping Array from ThermoFisher(thermofisher.com); CanineHD Whole-Genome Genotyping BeadChip fromIllumina (illumina.com). In some embodiments, the one or morepolynucleotides or oligonucleotides that specifically identify thepresence or absence of a retrogene insertion encoding canine fibroblastgrowth factor 4 (FGF4) on canine chromosome 12 can be further oradditionally attached to a canine genotyping array, e.g., an Axiom™Canine Genotyping Array from ThermoFisher (thermofisher.com) or aCanineHD Whole-Genome Genotyping BeadChip from Illumina. Constructionand use of microarrays is known in the art and described, e.g., inBowtell and Sambrook, “DNA Microarrays: A Molecular Cloning Manual,”Cold Spring Harbor Laboratory Press; 1st edition (Sep. 15, 2002). Insome embodiments, the solid support is a microbead.

4. Methods of Diagnosis

a. Obtaining a Biological Sample

The present diagnostic methods are useful for identifying whether acanine is genetically predisposed to suffer from skeletal dysplasia (SD)and/or intervertebral disc disease (IVDD) by determining the presence orabsence of a retrogene insertion encoding canine fibroblast growthfactor 4 (FGF4) on canine chromosome 12. The methods can involveobtaining a biological sample from a canine suspected of beinggenetically predisposed to suffer from skeletal dysplasia (SD) and/orintervertebral disc disease (IVDD).

The biological sample suitable for testing by the methods describedherein comprises a template nucleic acid, e.g., genomic DNA. Thebiological sample can include body fluids including whole blood, serum,plasma, cerebrospinal fluid, urine, lymph fluids, semen, and variousexternal secretions of the respiratory, intestinal and genitourinarytracts, tears, saliva, milk, white blood cells, myelomas, and the like;and biological fluids such as cell extracts, cell culture supernatants;fixed tissue specimens; and fixed cell specimens. Biological samples canalso be from solid tissue, including hair bulb, skin, muscle, biopsy orautopsy samples or frozen sections taken for histologic purposes. Thesesamples are well known in the art. A biological sample is obtained fromany canine to be tested for retrogene insertion encoding caninefibroblast growth factor 4 (FGF4) on canine chromosome 12 as describedherein. In some embodiments, the canine has lineage of a breed having apredisposition to chondrodystrophy (e.g., a chondrodystrophic (CD)breed, e.g., as reviewed in Smolders, et al., Vet J. 2013 March;195(3):292-9). Illustrative chondrodystrophic (CD) dog breeds includewithout limitation, e.g., American Cocker Spaniel, Basset Hound, Beagle,Cardigan Welsh Corgi, Chesapeake Bay Retriever, Chihuahua, Coton deTulear, Dachshund, English Springer Spaniel, French Bulldog, JackRussell Terrier, Miniature Schnauzer, Nova Scotia Duck TollingRetriever, Pekingese, Pembroke Welsh Corgi, Poodle, Portuguese WaterDog, Scottish Terrier, and Shih Tzu. A biological sample can besuspended or dissolved in liquid materials such as buffers, extractants,solvents and the like.

The biological sample may be obtained from a canine exhibiting symptomsof skeletal dysplasia (SD), chondrodystrophy and/or intervertebral discdisease (IVDD). In some embodiments, the canine is asymptomatic, but issuspected of being predisposed to developing skeletal dysplasia (SD),chondrodystrophy and/or intervertebral disc disease (IVDD), e.g., due tobreed, parentage or lineage. In some embodiments, the biological sampleis from a canine who has a parent, grandparent or sibling that is or hassuffered from skeletal dysplasia (SD), chondrodystrophy and/orintervertebral disc disease (IVDD). In certain embodiments, a biologicalsample is also obtained from an canine is not suffering from orsuspected of developing skeletal dysplasia (SD), chondrodystrophy and/orintervertebral disc disease (IVDD) as a negative control. In certainembodiments, a biological sample is also obtained from a canine known tobe suffering from skeletal dysplasia (SD), chondrodystrophy and/orintervertebral disc disease (IVDD) as a positive control.

b. Detecting the Genotype

The retrogene insert encoding canine fibroblast growth factor 4 (FGF4)on canine chromosome 12 (“CAF12 FGF4 insert”) can be detected using anymethods known in art, including without limitation amplification,sequencing and hybridization techniques. Detection techniques forevaluating nucleic acids for the presence of a single base changeinvolve procedures well known in the field of molecular genetics.Methods for amplifying nucleic acids find use in carrying out thepresent methods. Ample guidance for performing the methods is providedin the art. Exemplary references include manuals such as PCR Technology:PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION (ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992); PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS (eds. Innis, et al., Academic Press, San Diego, Calif.,1990); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, 1990-2017,including supplemental updates; Green and Sambrook, Molecular Cloning, ALaboratory Manual (4th Ed, 2012).

The nucleic acid template is isolated from the biological sample and aregion of the CAF12 FGF4 insert (e.g., the 5′- or 3′-ends) is amplifiedusing an oligonucleotide pair to form nucleic acid amplificationproducts of all or part of the CAF12 FGF4 insert, generally alsoincluding flanking or abutting sequences of canine chromosome 12.Amplification can be by any of a number of methods known to thoseskilled in the art including PCR, and the methods are intended toencompass any suitable techniques of DNA amplification. A number of DNAamplification techniques are suitable for use with the present methods.Conveniently such amplification techniques include methods such aspolymerase chain reaction (PCR), strand displacement amplification(SDA), nucleic acid sequence based amplification (NASBA), rolling circleamplification, T7 polymerase mediated amplification, T3 polymerasemediated amplification and SP6 polymerase mediated amplification. Theprecise method of DNA amplification is not intended to be limiting, andother methods not listed here will be apparent to those skilled in theart and their use is within the scope of the invention.

In some embodiments, the polymerase chain reaction (PCR) process is used(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR involves the useof a thermostable DNA polymerase, known sequences as primers, andheating cycles, which separate the replicating deoxyribonucleic acid(DNA), strands and exponentially amplify a gene of interest. Any type ofPCR, including quantitative PCR, RT-PCR, hot start PCR, LA-PCR,multiplex PCR, touchdown PCR, finds use. In some embodiments, real-timePCR is used.

The amplification products are then analyzed in order to detect thepresence or absence of the CAF12 FGF4 insert that is associated withcanine skeletal dysplasia (SD) and/or intervertebral disc disease(IVDD), as discussed herein. By practicing the methods of the presentmethods and analyzing the amplification products it is possible todetermine the genotype of individual canines with respect to the CAF12FGF4 insert.

In some embodiments, analysis may be made by restriction fragment lengthpolymorphism (RFLP) analysis of a PCR amplicon produced by amplificationof genomic DNA with the oligonucleotide pair. In order to simplifydetection of the amplification products and the restriction fragments,those of skill will appreciate that the amplified DNA will furthercomprise labeled moieties to permit detection of relatively smallamounts of product. A variety of moieties are well known to thoseskilled in the art and include such labeling tags as fluorescent,bioluminescent, chemiluminescent, and radioactive or colorigenicmoieties.

A variety of methods of detecting the presence and restriction digestionproperties of CAF12 FGF4 insert amplification products are also suitablefor use with the present methods. These can include methods such as gelelectrophoresis, mass spectroscopy or the like. The present methods arealso adapted to the use of single stranded DNA detection techniques suchas fluorescence resonance energy transfer (FRET). For FRET analysis,hybridization anchor and detection probes may be used to hybridize tothe amplification products. The probes sequences are selected such thatin the presence of the SNP, for example, the resulting hybridizationcomplex is more stable than if there is a G or C residue at a particularnucleotide position. By adjusting the hybridization conditions, it istherefore possible to distinguish between animals with the SNP and thosewithout. A variety of parameters well known to those skilled in the artcan be used to affect the ability of a hybridization complex to form.These include changes in temperature, ionic concentration, or theinclusion of chemical constituents like formamide that decrease complexstability. It is further possible to distinguish animals heterozygousfor the SNP versus those that are homozygous for the same. The method ofFRET analysis is well known to the art, and the conditions under whichthe presence or absence of the SNP would be detected by FRET are readilydeterminable.

Suitable sequence methods of detection also include e.g., dideoxysequencing-based methods and Maxam and Gilbert sequence (see, e.g.,Green and Sambrook, supra). Suitable HPLC-based analyses include, e.g.,denaturing HPLC (dHPLC) as described in e.g., Premstaller and Oefner,LC-GC Europe 1-9 (July 2002); Bennet et al., BMC Genetics 2:17 (2001);Schrimi et al., Biotechniques 28(4):740 (2000); and Nairz et al., PNASUSA 99(16):10575-10580 (2002); and ion-pair reversed phaseHPLC-electrospray ionization mass spectrometry (ICEMS) as described ine.g., Oberacher et al.; Hum. Mutat. 21(1):86 (2003). Other methods forcharacterizing retrogene inserts include, e.g., single base extensions(see, e.g., Kobayashi et al, Mol. Cell. Probes, 9:175-182, 1995);single-strand conformation polymorphism analysis, as described, e.g, inOrita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989), allelespecific oligonucleotide hybridization (ASO) (e.g., Stoneking et al.,Am. J. Hum. Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166,1986; EP 235,726; and WO 89/11548); and sequence-specific amplificationor primer extension methods as described in, for example, WO 93/22456;U.S. Pat. Nos. 5,137,806; 5,595,890; 5,639,611; and 4,851,331;5′-nuclease assays, as described in U.S. Pat. Nos. 5,210,015; 5,487,972;and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA88:7276-7280.

Methods for detecting single base changes well known in the art oftenentail one of several general protocols: hybridization usingsequence-specific oligonucleotides, primer extension, sequence-specificligation, sequencing, or electrophoretic separation techniques, e.g.,singled-stranded conformational polymorphism (SSCP) and heteroduplexanalysis. Exemplary assays include 5′ nuclease assays, template-directeddye-terminator incorporation, molecular beacon allele-specificoligonucleotide assays, single-base extension assays, and scoring byreal-time pyrophosphate sequences. Analysis of amplified sequences canbe performed using various technologies such as microchips, fluorescencepolarization assays, and matrix-assisted laser desorption ionization(MALDI) mass spectrometry. In addition to these frequently usedmethodologies for analysis of nucleic acid samples to detect single basechanges, any method known in the art can be used to detect the presenceof the CAF12 FGF4 insert described herein.

For example FRET analysis can be used as a method of detection.Conveniently, hybridization probes comprising an anchor and detectionprobe, the design of which art is well known to those skilled in the artof FRET analysis, are labeled with a detectable moiety, and then undersuitable conditions are hybridized a CAF12 FGF4 insert amplificationproduct containing the site of interest in order to form a hybridizationcomplex. A variety of parameters well known to those skilled in the artcan be used to affect the ability of a hybridization complex to form.These include changes in temperature, ionic concentration, or theinclusion of chemical constituents like formamide that decrease complexstability. The presence or absence of the CAF12 FGF4 insert is thendetermined by the stability of the hybridization complex. The parametersaffecting hybridization and FRET analysis are well known to thoseskilled in the art. The amplification products and hybridization probesdescribed herein are suitable for use with FRET analysis.

In one embodiment, the CAF12 FGF4 insert is detecting using one or moreoligonucleotide pairs configured or designed to detect the 5′-end and/orthe 3′-end of the retrogene insertion. In some embodiments, the one ormore oligonucleotide pairs are configured or designed or constructed todetect the 5′-end of the retrogene insertion located at nucleic acidresidue 1002 of SEQ ID NO:1. In some embodiments, an oligonucleotide inthe one or more oligonucleotide pairs hybridizes to a sequence segmentwithin nucleic acid residues 1-1001 of SEQ ID NO:1. In some embodiments,the one or more oligonucleotide pairs are configured to detect the3′-end of the retrogene insertion located at nucleic acid residue 1000of SEQ ID NO:2. In some embodiments, an oligonucleotide in the one ormore oligonucleotide pairs hybridizes to a sequence segment withinnucleic acid residues 1001-2000 of SEQ ID NO:2. In some embodiments, theone or more oligonucleotide pairs comprise one or more forward primersselected from the group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ IDNO:2), GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ IDNO:4), AGCCTGATGGCTGGACTGTA (SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQID NO:6) and one or more reverse primers selected from the groupconsisting of TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7),CCTGATTTTGAGACAGCCAAA (SEQ ID NO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9)and TGAGTGGGTTAAGGGTTTCG (SEQ ID NO:10). In some embodiments, the one ormore oligonucleotides comprise one or more forward primers selected fromthe group consisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2) andGTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and reverse primerTGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7).

c. Identifying or Selecting the Canine Based on Genotype

The methods identify individual canines based on the knowledge of thepresence or absence of a retrogene insertion encoding canine fibroblastgrowth factor 4 (FGF4) on chromosome 12. Presence of the CAF12 FGF4insert is statistically correlated with a predisposition to developcanine skeletal dysplasia (SD) and/or intervertebral disc disease (IVDD)in comparison to a canine that does not have the CAF12 FGF4 insert.

With the knowledge of the canine's genotype with respect to the CAF12FGF4 insert, one can then identify and sort canines into groups of likephenotype(s), or otherwise use the knowledge of the genotype in order topredict which canines will have the desired phenotypes, for example,decreased susceptibility to develop SD and/or IVDD. Knowledge of thecanine's genotype with respect to the CAF12 FGF4 insert allows a breederto encourage breeding between canines with a desired CAF12 FGF4 genotype(e.g., where the CAF12 FGF4 insert is absent), and to discouragebreeding between canines with an undesirable CAF12 FGF4 genotype (e.g.,where the CAF12 FGF4 insert is present).

Selecting or sorting can be taken to mean placing canines in physicalgroupings such as pens, so that canines of like genotype are keptseparate from canines of a different genotype. This would be a usefulpractice in the case of breeding programs where it would be desirable toproduce canines of particular genotypes. For example, it may bedesirable to breed canines that do not have the CAF12 FGF4 insert, suchthat breeding among these canines would only produce canines with adesired genotype with respect to the CAF12 FGF4 insert. On the otherhand, it may also be desirable to decrease production of animals with anundesired CAF12 FGF4 insert genotype. Separating out canines with thedesired CAF12 FGF4 insert genotype(s) would prevent canines with anundesired CAF12 FGF4 insert genotype from breeding with caninespossessing a desired CAF12 FGF4 insert genotype, facilitating thereproduction of canines with an increased susceptibility to develop SDand/or IVDD, which is associated with presence of the CAF12 FGF4 insert.Furthermore, ensuring that at least one canine in a breeding pairpossesses desired CAF12 FGF4 insert genotype allows for the frequency ofthe desired CAF12 FGF4 insert genotype to be increased in the next, andsubsequent generations.

Sorting may also be of a “virtual” nature, such that a canine's genotypeis recorded either in a notebook or computer database. In this case,canines could then be selected based on their known genotype without theneed for physical separation. This would allow one to select for caninesof desired phenotype where physical separation is not required. Forexample, many canine breed registries perform parentage verificationusing a set of alleles each time a canine is registered.

5. Kits

Further provided are kits. In some embodiments, the kits comprise one ormore oligonucleotide pairs configured or designed to detect the presenceor absence of a retrogene insertion encoding canine fibroblast growthfactor 4 (FGF4) on chromosome 12. In some embodiments, theoligonucleotide pairs detect the 5′-end and/or the 3′-end of theretrogene insertion. In some embodiments, the 5′-end of the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) comprises anucleic acid sequence having at least 90% sequence identity, e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, to nucleic acid residues 1002-2001 SEQ ID NO:1. In someembodiments, the one or more oligonucleotide pairs are configured todetect the 5′-end of the retrogene insertion located at nucleic acidresidue 1002 of SEQ ID NO:1. In some embodiments, an oligonucleotide inthe one or more oligonucleotide pairs hybridizes to a sequence segmentwithin nucleic acid residues 1-1001 of SEQ ID NO:1. In some embodiments,the 3′-end of the retrogene insertion encoding canine fibroblast growthfactor 4 (FGF4) comprises a nucleic acid sequence having at least 90%sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, to nucleic acid residues 1-1000 SEQID NO:2. In some embodiments, the one or more oligonucleotide pairs areconfigured to detect the 3′-end of the retrogene insertion located atnucleic acid residue 1000 of SEQ ID NO:2. In some embodiments, anoligonucleotide in the one or more oligonucleotide pairs hybridizes to asequence segment within nucleic acid residues 1001-2000 of SEQ ID NO:2.In some embodiments, the one or more oligonucleotide pairs comprise oneor more forward primers selected from the group consisting of:ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2), GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3),CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4), AGCCTGATGGCTGGACTGTA (SEQ ID NO:5)and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and one or more reverse primersselected from the group consisting of TGCTGTAGATTTTGAGGTGTCTT (SEQ IDNO:7), CCTGATTTTGAGACAGCCAAA (SEQ ID NO:8), TTGATGCCCAGGAGGTAGTC (SEQ IDNO:9) and TGAGTGGGTTAAGGGTTTCG (SEQ ID NO:10). In addition, the kit cancomprise appropriate buffers, salts and other reagents to facilitateamplification and/or detection reactions (e.g., primers, labels,secondary antibodies).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

FGF4 retrogene on CFA12 is responsible for chondrodystrophy andintervertebral disc disease in dogs

Materials and Methods

Phenotype and Sample Collection. Blood samples, height at the withers,thoracic limb radiographs, and pictures (when possible), were collectedfrom privately owned NSDTRs affected with owner or veterinarian reportedskeletal dysplasia (SD), as well as phenotypically “normal” dogs.Additionally, blood samples were collected from cases of type Iintervertebral disc disease (IVDD) seen at the University of California,Davis School of Veterinary Medicine Teaching Hospital and privatelyowned NSDTRs. IVDD cases were defined by the presence of one or moremineralized thoracolumbar intervertebral discs (IVD), as confirmed byvertebral column radiographs and/or the presence of extruded calcifieddegenerative disc material at surgery or necropsy. DNA was extractedfrom EDTA whole blood samples using Gentra Puregene DNA purificationextraction kit (Qiagen, Valencia, Calif.). Collection of canine sampleswas approved by the University of California, Davis Animal Care and UseCommittee (protocol #18561).

Genome-wide Association Study (GWAS). Genome-wide single nucleotidepolymorphism (SNP) genotyping was performed using the Illumina Canine HD174,000 SNP array (Illumina, San Diego, Calif.) for 13 NSDTR SD casesand 15 NSDTR controls with no reported SD. SNPs were pruned fromanalysis if the minor allele frequency was <5% and the call rate <90%.Additionally, a separate GWAS was performed for 36 IVDD cases from 16breeds and 31 controls with no reported IVDD from 14 breeds (number ofdogs from each breed listed in Table 1). The SNPs were pruned fromanalysis if the minor allele frequency was <10% and the call rate <90%.Chi-square association analysis, Bonferroni adjustments, and genomicinflation calculations were performed in Plink (59).

TABLE 1 Number of dogs per breed used in across breed IVDD GWAS # ofBreed Dogs Phenotype Basset Hound 1 Case Beagle 1 Case Boston Terrier 3Control Brittany 3 Control Bulldog 1 Control Cardigan Welsh Corgi 1 CaseChihuahua 3 Case Collie 2 Control Coton de Tulear 12 6 Cases, 6 ControlsDachshund 4 Case Dalmatian 1 Control French Bulldog 3 Case lbizan Hound2 Control Jack Russell Terrier 1 Control Lacy Dog 1 Control Maltese 1Case Miniature Poodle 1 Case Mix 15 11 Cases, 4 Controls NSDTR 1 CasePembroke Welsh Corgi 1 Case Poodle 1 Control Rottweiler 1 Case ShetlandSheepdog 2 Control Shih Tzu 1 Case Whippet 3 Control Yorkshire Terrier 1Control

Whole Genome Sequencing (WGS). For library prep and sequencing, DNA wasfragmented using the Covaris E220 sonicator (Covaris Inc.), and thenfollowed by selection of 550 bp insert size fragments. Illuminapaired-end 150 bp libraries were prepared using PCR-free library prepkits. Sequencing was done on the HiSeq2500 platform at the BGIsequencing facility. Reads were scanned for sequencing adaptors and lowquality sequences using the Trimmomatic software package (V 0.36) 60.High quality reads were aligned to the dog reference genome canFam3 (61)using the BWA-MEM algorithm of the BWA software package (v0.7.7) (62).Duplicate reads were excluded using Picard v2.2.4 tool MarkDuplicates(63). Variant calling was performed using GATK HaplotypeCaller (v3.5)(64). SNPs and small insertions and deletions (indels) were investigatedfor segregation with the IVDD phenotype in a 4 Mb region(chr12:33.1-35.5 Mb (canFam3)), which included the critical intervalidentified using GWAS. Segregation of variants was performed using 2cases (1 affected NSDTR and 1 Dachshund) compared to 83 controls ofvarious normal legged breeds. To investigate the presence of largeindels within the critical interval, BAM files covering the associatedinterval were scanned by eye using the Integrative Genomics Viewer (IGV,Broad Institute) in 2 cases (1 Dachshund and 1 SD affected NSDTR) and 2controls (1 NSDTR and 1 Saluki) for segregation with the IVDD phenotype.The reads were viewed as color-coded by insert size and pair orientationto flag mate pairs that mapped to other chromosomes in order to easilyidentify large insertions and deletions.

Investigation of Large Indels. BAM files for additional control genomes(1 NSDTR, 1 Weimaraner, 1 Border Collie(ncbi.nlm.nih.gov/biosample/SAMN03801652)) were used to evaluatesegregation of the 8 identified large indels. The remaining segregatinglarge indels (Deletions 1-3 and Insert 5) were investigated inadditional cases and controls using PCR (primers listed in Table 2).

TABLE 2Primers used to large indels identified CFA12: 33.1-35.5 Mb (canFam3)Forward Primer (5′→3′) Reverse Primer (5′→3′) CFA7 InsertCTCTGTGGACCTCTTTCAACG TGACACCAGTGAGAATTGCAT Deletion 1TGCTTGCTCCAGCTCTGTTA TTGGCCATAATTTTCCTTGG Deletion 2AAATGGCATATGGGCTGAGT TCTGCAAAACAGCTTGCATT Deletion 3CACTGTTGGCAGTCCTCAAA AAAGCCGGTTGTTGATGAAG Insert 5 ATGCTACACCACTCCCTGCTATCCTTGCCAAAACTGATGG

Investigation of potential insert from CFA 7. The integrity of theregion on CFA7 (approximately chr7:68,371,500-68,374,000) was testedusing PCR and sequencing, as read mates at the FGF4 insertion site onCFA12 also mapped to this location. Primers (listed in Table 2) spanningthe potentially inserted segment of CFA7 were used in PCR for 6 cases (1NSDTR, 1 Beagle, 1 Basset Hound, 1 French Bulldog, and 1 Maltese) and 1control (Boston Terrier) using the LongAmp Taq PCR Kit (New EnglandBiolabs, Ipswich, Mass., USA). If the genome assembly is correct, PCRproduct size should be 2,077 bp, if not, the product would only be 842bp. PCR products for 3 cases and 1 control were sequenced on an AppliedBiosystems 3500 Genetic Analyzer using the Big Dye Terminator SequencingKit (Life Technologies, Burlington, ON, Canada) and the products alignedto the UCSC genome browser using BLAT (genome.ucsc.edu/).

Cloning. To obtain the full FGF4 retrogene insertion sequence on CFA12,as well as on CFA18 for comparison, the PCR products using CFA12 FGF4Insertion and CFA18 FGF4 Insertion primer pairs (Table 3), respectively,were cloned using the TOPO TA Cloning kit with PCR2.1 TOPO (ThermoFisher Scientific, Inc., Waltham, Mass., USA) and One Shot TOP10competent cells. CFA12 and 18 FGF4 insertions were amplified fromgenomic DNA using the LongAmp Taq PCR Kit (New England Biolabs, Ipswich,Mass., USA) using primers flanking the inserts on CFA12 and 18,respectively and recommended cycling conditions. The CFA12 FGF4insertion was cloned from a Beagle and the CFA18 FGF4 insertion wascloned from a Dachshund. Plasmid DNA was extracted using the QIAprepSpin Miniprep Kit (Qiagen, Valencia, Calif., USA). To confirm successfultransformation, plasmid DNA was sequenced using vector primers M13.F andM13.R (Thermo Fisher Scientific, Inc., Waltham, Mass., USA) on anApplied Biosystems 3500 Genetic Analyzer using the Big Dye TerminatorSequencing Kit (Life Technologies, Burlington, ON, Canada) and analyzedusing VectorNTI software (Thermo Fisher Scientific, Inc., Waltham,Mass., USA). Internal FGF4 primers were also used to create overlappingcontigs and ensure the entire insert was sequenced (listed in Table 3).CFA12 and 18 FGF4 inserts were aligned to endogenous FGF4 sequence toconfirm presence of the gene's entire coding sequence. Polymorphismsidentified were not queried in additional dogs, so could be due tosequencing or cloning error or be dog/breed specific.

TABLE 3Primers used to sequence and assay the FGF4 insert on CFA12 and 18Forward Primer (5′→3′) Reverse Primer (5′→3′) CFA12 FGF4 InsertionACAGCTGGCATGGTCAGTTA CCTGATTTTGAGACAGCCAAA CFA18 FGF4 InsertionTTGGGAATGTCAAACCACTG AGGGCCAAGTGTCCAATACA FGF4.F1 GTGTTTGCATGGAGGAAGGTFGF4.F2 CTGAGCAAGAACGGGAAGAC FGF4.F3 AGCCTGATGGCTGGACTGTA FGF4.F4GTCCGTGCGGTGAAATAAAA FGF4.R1 TTGATGCCCAGGAGGTAGTC FGF4.R2TGAGTGGGTTAAGGGTTTCG FGF4_TSS.F1 GCGCTCCGCACCGAGTCC FGF4_TSS.F2CTCCATGCAGCCCGGGTA

Transcription start site (TSS) Investigation. To ensure the CFA12 FGF4insertion included the transcription start site (TSS), PCR was performedusing cDNA with primers at varying positions 5′ to the FGF4 start codon(Table 3). cDNA was synthesized from RNA extracted from neonatalintervertebral disc (IVD) and vertebral body (VB), as described below,from a Beagle. Amplified products were visualized on a 2% agarose gel.

Genotyping Assay. The presence or absence of the FGF4 insertion on CFA12and 18 was assayed using a PCR-based genotyping test. Three primer PCRwas performed using a forward and reverse primer flanking the respectiveinsert, as well an additional forward primer located within the FGF4insert (listed in Table 4). For the CFA12 FGF4 insert assay, eachreaction included 12 μl water, 2 μl 10× Buffer, 0.8 μl 25 mM MgCl₂, 2 μldNTP, 0.5 μl of the external forward primer (20 μM), 0.7 μl of theinternal forward primer (20 μM), 0.8 μl of the reverse primer (20 μM),0.2 μl of HotStarTaq DNA Polymerase (Qiagen, Valenica, Calif., USA), and1 μl of DNA. For the CFA18 FGF4 insert assay, each reaction included 12μl water, 2 μl 10× Buffer, 0.8 μl 25 mM MgCl₂, 2 μl dNTP, 0.5 μl of theexternal forward primer (20 μM), 0.6 μl of the internal forward primer(20 μM), 0.9 μl of the reverse primer (20 μM), 0.2 μl of HotStarTaq DNAPolymerase (Qiagen, Valenica, Calif., USA), and 1 μl of DNA. Amplifiedproducts were visualized on a 2% agarose gel. For the CFA18 FGF4insertion: wild type dogs had a single 388 bp band, homozygous mutantsamples had a single 168 bp band, and both bands were present inheterozygous samples. For the CFA12 FGF4 insertion, a single 333 bp bandwas present in wild type samples, a single 654 bp band was present inthe homozygous mutant samples, and both bands were present inheterozygous samples.

TABLE 4 External Internal External Forward Forward Reverse Primer PrimerPrimer (5′→3′) (5′→3′) (5′→3′) CFA12 ACAGCTGGCAT GTCCGTGCGGTGATGCTGTAGATTTTG FGF4 GGTCAGTTA AATAAAA AGGTGTCTT Insert Genotyping AssayCFA18 TTGGGAATGTC GTCCGTGCGGTGA GTTCCCTCCATTTC FGF4 AAACCACTG AATAAAAGGTTT Insert Genotyping Assay

Height at the withers was collected for 20 male NSDTRs to associate dogheight with the CFA12 FGF4 insertion. Significance was determined usinga one-tailed T-test and a threshold cutoff of p <0.05.

To compare the significance of association of the CFA12 FGF4 insertionto the most highly associated SNP and the CFA18 FGF4 insertion, 34 of 36IVDD cases and 31 controls were genotyped for both the CFA12 and CFA18FGF4 insertions. Chi square and odds ratio analysis was performed inPlink (59)

IVDD Incidence Across Breeds seen at UC Davis SVM VMTH. To assessfrequency of IVDD in specific breeds, UC Davis School of VeterinaryMedicine Teaching Hospital (VMTH) records were searched between 1980 and2016 for dogs with a clinical diagnosis of “disc/k disease” or “IVDD.”Significantly over or underrepresented breeds relative to the total VMTHhospital population were determined based on a Chi-squared test.Significance was set at p <0.05.

qRT-PCR. cDNA was prepared from RNA that was extracted from IVD or VBdissected from neonatal canine tail samples, skeletal muscle, andtestis, using the QuantiTect Reverse Transcription Kit (Qiagen,Valencia, Calif., USA) and the RNeasy Fibrous Tissue Mini Kit (Qiagen,Valencia, Calif., USA), respectively. Additionally, cDNA was made fromcommercially available Beagle skeletal muscle and testis RNA (Zyagen,San Diego, Calif., USA).

Semi-quantitative RT-PCR was performed for genes within and near thecritical interval, including COL9A1, SMAP1, B3GAT2, OGFRL1, LINC00472,RIMS1, KCNQ5, and COL12A1, as well as FGF4, for VB, IVD, skeletalmuscle, and testis cDNA from a case and control. RPS5 was also includedas a housekeeping gene control. All case samples were collected from aBeagle, while control VB and IVD were from a Cane Corso and skeletalmuscle and testis from a Labrador Retriever. Primers spanning at least 1intron were designed for all genes using Primer3, except for RPS5 inwhich the primers were as recommended by Brinkhof et al. (65,66). Eachreaction included 13.9 μl water, 2 μl 10× Buffer with MgCl₂, 1 μl dNTP,1 μl of each forward and reverse primers (20 μM) (listed in Table 5), 1μl of HotStarTaq DNA Polymerase (Qiagen, Valenica, Calif., USA), and 1μl of cDNA made from 1000 ng of RNA. Amplified products were visualizedon a 2% agarose gel.

TABLE 5 Primers used for semi-qPCR and qRT-PCR experimentsForward Primer Reverse Primer (5′→3′) (5′→3′) COL9A1TTCTGTCGGACCAAGAGGAC CCATCATGAAAGCCAATGGT SMAP1 AGGCTCAGAAGCTGAACGAGTCTGGTGTCCATTGGTCTAG G B3GAT2 CCTACAGCCTGGAGCTGTTC GGCTTTTGGATTGGACAAGAOGFRL1 AAGCAACTGCCAAACCAAAG GTTCTCTCAGGGGGAAAAGC LINC00472GGGCTGTACTGGCTCATTGT AGAGCAGCACACCCAAGTCT RIMS1 TGGCCATCTCTGCTCCTACTACCTCAGAACCAGCACCTGT KCNQ5 TTGTGGAAAAGGATGCCAAT GGCGGTGCTGTTCTTGTACTCOL12A1 CTACAGGGGACGACAGAAGG CTGCTTCTGCTCTGGTGAGA FGF4GACTACCTCCTGGGCATCAA GTCTTCCCGTTCTTGCTCAG RPS5 TCACTGGTGAGAACCCCCTCCTGATTCACACGGCGTAG

Quantitative RT-PCR was performed for FGF4 using cDNA synthesized from500 ng of RNA extracted from IVD and VB dissected from the tails of 4cases (4 Beagle) and 5 controls (1 Rottweiler and 4 Cane Corso). A2-step cycle protocol was employed using the Rotor-Gene SYBR Green PCRKit (Qiagen, Valencia, Calif., USA) on the Rotor Gene Q real-time PCRsystem: Initial denaturation at 95° C. for 5 minutes; Annealing at 95°C. for 5 seconds and extension at 60° C. for 10 seconds for 35 cycles;Final melt curve. Samples were run in triplicate with 20 ng of templatecDNA each for both the IVD and VB experiment. FGF4 transcript levelswere normalized to RPS5 and analyzed for fold change differences inexpression using ΔΔCT. A technical replicate was removed from analysisif the standard deviation of the 3 technical replicates was greater than1, and a sample was removed from analysis if there were less than 2technical replicates that met this criteria. Fold change in expressionof FGF4 in IVD and VB was calculated by taking 2^(−(ΔΔCT)) for eachtissue, respectively. Statistical significance was assessed using aMann-Whitney-Wilcoxin test.

Results

A form of skeletal dysplasia (SD) is common in the NSDTR and ischaracterized by variable decrease in limb length and associatedabnormalities including long bone bowing, physeal widening, and jointincongruity. (FIG. 1a, 1b ). On physical examination, in addition toshorter limbs, SD dogs may also have valgus limb deformities and largerears (pinnae). While SD is a common phenotype in the breed, the degreeof severity is highly variable.

To determine a region of the genome associated with SD in the NSDTR,genome-wide association analysis was performed using 13 NSDTR withsevere SD and 15 NSDTR controls without severe SD. There were 41 SNPsthat were genome-wide significant with a pBonferroni <0.05, all presentbetween chr12:35,413,695-46,117,273 (top SNP-chr12:36,790,324pBonferroni=0.007232) (canFam2) (FIG. 1c , FIG. 2).

Underlying this strong association for SD in NSDTRs was an approximately12 Mb critical interval from chr12:36-48 Mb (canFam2). Since the NSDTRSD phenotype is not uncommon in different dog breeds, we investigatedhaplotype sharing across breeds and observed that a portion of thisassociated haplotype was shared with two breeds of dog consideredclassically chondrodystrophic: the American Cocker Spaniel and Beagle(1,3). By plotting the minor allele frequency (MAF) across this intervalfor 7 American Cocker Spaniels, 14 Beagles, and 13 SD affected NSDTR,the critical interval identified via GWAS for SD was shortened to ashared haplotype from chr12:36.4-38.3 Mb (canFam2) (FIG. 3a ).Interestingly, the breeds that shared this smaller haplotype have beenwell characterized as chondrodystrophic and predisposed to Hansen's typeI IVDD, suggesting that the associated SD locus in the NSDTR may also becausing chondordystrophy across dog breeds.

In order to test this hypothesis, a second genome-wide association studywas performed using IVDD affected cases (n=36) and unaffected controls(n=31) across 26 dog breeds (Table 6). The most highly associated SNPwas located on CFA12 (chr12:36,909,311 (canFam2)) with ap_(raw)=3.191×10⁻¹⁵, pBonferroni=4.02×10⁻¹⁰, and odds ratio of 32.67(FIG. 3b ). Observing LD with the highest associated SNP using r²values, the critical interval identified via GWAS for IVDD overlaps withthat seen when mapping MAF across breeds (FIG. 3c ).

TABLE 6 CFA12 Increase FGF4 Insert VMTH IVDD Chi or Allele Breed Pop. %% square Significance Decrease Frequency American Cocker Spaniel 2.32.99 9.15 0.003 Increase 0.95 Basset Hound 0.51 1.24 45.21 1.77 × 10⁻¹¹Increase 0.68 Beagle 0.86 2.42 120.69 4.47 × 10⁻²⁸ Increase 0.97 Corgi0.67 1.82 85.41  2.43 × 10−²⁰ Increase 0.82 Dachshund 2.68 25.95 8874.25 <0.000001 Increase 0.98 French Bulldog 0.39 1.89 248.09 6.77 × 10⁻⁵⁶Increase 0.94 Pekingese 0.35 1.6 189.72 3.66 × 10⁻⁴³ Increase 0.44Brittany 0.49 0.24 5.34 0.021 Decrease 0.00 Bulldog 0.97 0.07 35.83 2.16× 10⁻⁹  Decrease 0.00 Cairn Terrier 0.23 0.07 4.46 0.035 Decrease 0.00Scottish Terrier 0.31 0.1 6.18 0.013 Decrease 0.05 Shetland Sheepdog0.93 0.19 25.29 4.95 × 10⁻⁷  Decrease 0.00 Springer Spaniel 0.96 0.625.11 0.024 Decrease 0.11 West Highland White Terrier 0.53 0.12 13.72 0.0002 Decrease 0.00 Yorkshire Terrier 1.56 0.96 10.13 0.002 Decrease0.00 Investigation of IVDD in breeds seen at the UC Davis School ofVeterinary Medicine Teaching Hospital: Canine cases seen at the UC DavisSchool of Veterinary Medicine Teaching Hospital between 1980 and 2016were queried for a clinical diagnosis of “disc/k disease” or “IVDD.”203,958 cases were seen, of which 4,177 were diagnosed with “disc/kdisease” or “IVDD.” The breeds shown have a p-value associated with anincrease or decrease in incidence of IVDD. Allele frequencies calculatedfrom on dogs genotyped in Table 10, below.

To identify a causative variant for SD and IVDD, paired-end whole genomesequences of 2 cases, 1 SD affected NSDTR and 1 IVDD affected Dachshund,and 83 unaffected controls were investigated in the associated interval.There were 9,156 SNP variants and 7,877 insertion/deletion (indel)variants identified from chr12:33.1-35.5 Mb (canFam3) (chr12:36.1-38.5Mb (canFam2)); however, none segregated with the IVDD phenotype. Thesame interval was also investigated by visual inspection of BAM files toflag mate pairs with unusual insert sizes in an effort to identify anylarge indels. Using the 2 cases and 2 controls, 8 large indels (>200 bp)were identified within the interval (Table 7). Four large indels did notsegregate when investigated in additional control genomes, while theremaining 4 were eliminated after PCR showed lack of segregation betweencases and controls.

TABLE 7 Indel Coordinates (canFam3) Indel Method of EliminationChr12:33,927,660-33,928,003 Deletion 1 PCR Chr12:34,256,430-34,256,530Deletion 2 PCR Chr12:34,467,000 Insertion 1 Additional GenomesChr12:34,734,000 Insertion 2 Additional Genomes Chr12:34,758,000Insertion 3 Additional Genomes Chr12:34,947,000 Insertion 4 AdditionalGenomes Chr12:35,228,600-35,228,800 Deletion 3 PCR Chr12:35:498,000Insertion 5 PCR Large indels identified via BAM file investigation:Coordinates and method of elimination for each of the 8 segregatinglarge indels identified in 2 cases (1 Dachshund and 1 SD NSDTR) and 2controls (1 NSDTR and 1 Saluki). Insertions 1-4 were eliminated based onlack of segregation with investigation of additional control genome BAMfiles. Deletions 1-3 and Insert 5 were eliminated based on lack ofsegregation demonstrated via PCR of additional cases and controls.

Visual inspection of the BAM files for read-pairs mapping to a differentchromosome location identified a region, located at approximatelychr12:33,710,200 (canFam3), that segregated with the 2 cases and 2controls (FIG. 4). At this location, read mates mapped to chr18:48.4 Mb(canFam3) and chr7:68.3 Mb (canFam3) in the NSDTR and Dachshund cases,but none of the controls. The reads that mapped to CFA18 aligned toendogenous FGF4, which was highly suggestive of a FGF4 retrogeneinsertion at this location. The reads that mapped to CFA7 wereinvestigated by PCR and appear to mark a genome assembly error or amutation within the dog used for the genome assembly (canFam3).

To investigate the potential FGF4 insert on CFA12, the region was PCRamplified using primers flanking the insertion site in an IVDD affectedBeagle. Wild type dogs without the insert had a single 615 bp band,while dogs homozygous for the CFA12 FGF4 insertion had an approximately4 kb product. Sanger sequencing showed the insertion on CFA12 is 3,209bp long (GenBank Accession # MF040221) and includes endogenous FGF4 cDNA(i.e. FGF4 exons spliced without introns), as shown in the insertschematic comparing endogenous FGF4 to the CFA12 insert (FIG. 5). Theinsert also contains a majority of the predicted 5′UTR, which includesthe transcription start site (TSS) as only PCR primers FGF4_TSS.F1 andFGF4.R1 yielded a product in RT-PCR using cDNA from neonatal Beagle IVD(Table 2).

In order to compare the CFA12 FGF4 retrogene to the previouslyidentified CFA18 FGF4 retrogene, it was necessary to obtain the fulllength sequence of the CFA18 insertion (26). The cloned product wassequenced using the flanking and common internal primers (Table 2),yielding a 2,665 bp insert (GenBank Accession # MF040222). While itcontained the same length 5′UTR and FGF4 cDNA as that seen in the CFA12FGF4 insert, the 3′UTR was shortened in comparison. The 3′UTR of theCFA18 FGF4 insert was followed by a sequence containing 30 adenine and 1guanine residues and a different target site duplication (TSD) sequence(AAG TCA GAC AGA G).

In order to assay the insertions in additional dogs, insertion andallele specific PCR based genotyping assays were developed for both theCFA12 FGF4 insertion and the previously identified CFA18 FGF4 insertion(FIG. 6a ). Twelve SD NSDTR cases from the GWAS were genotyped and werehomozygous for the CFA12 FGF4 insertion, while all controls wereheterozygous or wild type. Additionally, IVDD cases (n=7) from the NSDTRbreed were collected and were either homozygous mutant or heterozygousfor the CFA12 FGF4 insertion (Table 8). All NSDTR tested for the CFA18FGF4 insertion (n=31) were wild type, including SD and IVDD cases.NSDTRs with known height (n=20 males) at the withers were also genotypedfor the CFA12 FGF4 insertion to investigate the association of heightwith genotype status. Height and genotype were significantly associatedin a dose dependent manner when comparing wild type, heterozygous, andhomozygous dogs (FIG. 6b ).

TABLE 8 CFA12 FGF4 insert genotyping results for an additional 40 IVDDcases Breed Wild type Heterozygous Mutant Bichon Frise 0 1 2 Chihuahua 02 0 Dachshund 0 0 17 Dandie Dinmont 0 1 0 Terrier Mix 0 6 4 Nova ScotiaDuck 0 5 2 Tolling Retriever

To assess the significance of association of the CFA12 FGF4 insertionwith IVDD across breeds, dogs used in the IVDD GWAS were genotyped forboth insertions. All dogs' genotypes were concordant with phenotypeexcept for one case, a Rottweiler (Table 9). When associated with IVDD,the CFA12 FGF4 insertion was more highly associated than both the mosthighly associated SNP from the GWAS, as well as the CFA18 FGF4 insertion(FIG. 6c ). To further investigate the association of the CFA12 FGF4insertion with IVDD, 33 additional cases were genotyped for the CFA12FGF4 insertion: 10 were heterozygous and 23 were homozygous for the CFA12 FGF4 insertion (Table 8).

TABLE 9 CFA18 and CFA12 FGF4 insert genotype for 34 IVDD cases and 31controls used in the across breed IVDD GWAS Breed of Dog Case or CFA18FGF4 Insert Genotype CFA12 FGF4 Insert Genotype Genotyped Control WTHeterozygous Mutant WT Heterozygous Mutant Basset Hound Case (n = 1) 0 01 0 0 1 Beagle Case (n = 1) 1 0 0 0 0 1 Cardigan Welsh Case (n = 1) 0 10 0 1 0 Corgi Chihuahua Case (n = 3) 1 2 0 0 3 0 Coton de Tulear Case (n= 6) 0 2 4 0 1 5 Dachshund Case (n = 3) 0 0 3 0 0 3 French Bulldog Case(n = 3) 3 0 0 0 0 3 Maltese Case (n = 1) 0 0 1 0 0 1 Mix Case (n = 10) 14 5 0 6 4 NSDTR Case (n = 1) 1 0 0 0 1 0 Pembroke Welsh Case (n = 1) 0 01 0 0 1 Corgi Miniature Poodle Case (n = 1) 0 1 0 0 0 1 Rottweiler Case(n = 1) 1 0 0 1 0 0 Shih Tzu Case (n = 1) 0 0 1 0 1 0 Boston TerrierControl (n = 3) 3 0 0 3 0 0 Brittany Control (n = 3) 3 0 0 3 0 0 BulldogControl (n = 1) 1 0 0 1 0 0 Collie Control (n = 2) 2 0 0 2 0 0 Coton deTulear Control (n = 6) 0 0 6 2 4 0 Dalmatian Control (n = 1) 1 0 0 1 0 0Ibizan Hound Control (n = 2) 2 0 0 2 0 0 Jack Russel Control (n = 1) 1 00 1 0 0 Terrier Lacy Dog Control (n = 1) 1 0 0 1 0 0 Poodle Control (n= 1) 1 0 0 1 0 0 Shetland Control (n = 2) 2 0 0 2 0 0 Sheepdog WhippetControl (n = 3) 3 0 0 3 0 0 Mix Control (n = 4) 4 0 0 4 0 0 YorkshireTerrier Control (n = 1) 0 1 0 1 0 0

In order to investigate the breed distribution of the retrogeneinsertion, 568 dogs from 50 breeds were genotyped (Table 10). The CFA12FGF4 insertion segregates in the majority of breeds where it occurs andis present in small and medium sized dog breeds with high frequency(FIG. 7). Interestingly, all of the dogs with the CFA12 FGF4 insertionalso have large external ears (pinnae), which is consistent with thephenotype seen in the NSDTR.

TABLE 10 Genotyping results for 568 dogs from 50 different breeds forCFA12 FGF4 insertion. Breeds listed in bold showed segregation BreedWild type Heterozygous Mutant American Cocker Spaniel 0 1 9 AustralianCattle Dog 10 0 0 Australian Shepherd 10 0 0 Basset Hound 1 5 5 Beagle 01 17 Bernese Mountain Dog 10 0 0 Boston Terrier 3 0 0 Brittany 14 0 0Bulldog 11 0 0 Cairn Terrier 9 0 0 Cane Corso 5 0 0 Cardigan Welsh Corgi1 2 5 Cavalier King Charles Spaniel 0 0 9 Chesapeake Bay Retriever 29 70 Chihuahua 5 6 2 Collie 2 0 0 Coton de Tulear 2 5 5 Dachshund 0 1 27Dalmatian 1 0 0 Doberman Pinscher 15 0 0 English Springer Spaniel 7 2 0Fox Terrier 10 0 0 French Bulldog 0 4 28 German Shepherd Dog 10 0 0Golden Retriever 10 0 0 Great Dane 10 0 0 Ibizan Hound 3 0 0 IrishSetter 8 0 0 Jack Russell Terrier 1 1 9 Labrador Retriever 10 0 0 LacyDog 1 0 0 Maltese 0 0 1 Miniature Schnauzer 9 1 0 Mix 4 6 4 Newfoundland14 0 0 Nova Scotia Duck Tolling Retriever 6 15 12 Pekingese 2 5 1Pembroke Welsh Corgi 0 2 7 Poodle 4 7 4 Portuguese Water Dog 8 1 0Rottweiler 11 0 0 Scottish Terrier 9 1 0 Shetland Sheepdog 12 0 0 ShihTzu 3 8 1 Siberian Husky 10 0 0 Saint Bernard 10 0 0 Weimaraner 10 0 0West Highland White Terrier 10 0 0 Whippet 3 0 0 Yorkshire Terrier 11 00

Based on occurrence of IVDD at the Pritchard Veterinary MedicineTeaching Hospital at UC Davis, the breeds with a statistically higherfrequency of IVDD are also those with a higher frequency of the CFA12FGF4 insert allele, while the breeds with a statistically lowerfrequency of IVDD are those with a lower frequency of the CFA12 FGF4insert allele (Table 6).

To investigate the gene expression environment in which FGF4 inserted onCFA12, semi-quantitative RT-PCR was performed for genes across the IVDDassociated interval. Using cDNA derived from neonatal vertebral body(VB) and IVD, skeletal muscle, and testis, expression levels of genesacross the CFA12 associated interval were assayed in a Beagle case andCane Corso or Labrador Retriever control, including: COL9A1, SMAP1,B3GAT2, OGFRL1, LINC00472, RIMS1, KCNQ5, and COL12A1. Expressiondifferences between case and control were not apparent in these genes;however, we confirmed that all except RIMS1 are expressed in bothneonatal VB and IVD, supporting that FGF4 inserted itself in a genemilieu conducive to expression in IVD. Semi-q PCR for total FGF4(endogenous and retrogene products) in the same tissues showed increasedexpression across all tested tissue types in the case versus the control(FIG. 8).

In order to evaluate the effect of the CFA12 FGF4 retrogene insertion onoverall FGF4 transcript levels, quantitative RT-PCR was performed. Acomparison between samples homozygous for the CFA12 FGF4 insertion andsamples with only the endogenous copy of FGF4 (i.e. wild type for boththe CFA12 and CFA18 FGF4 insertions) showed a 19.47× higher (p=0.02857)and 2.16× higher (p=0.02857) expression of FGF4 in neonatal IVD and VB,respectively (FIG. 9).

Discussion

In this study, we report the identification of a FGF4 retrogeneinsertion in the dog genome responsible for chondrodystrophy across dogbreeds, characterized by both short limbs and susceptibility to Hansen'stype I intervertebral disc disease. A region was identified on CFA12 dueto association with a segregating form of skeletal dysplasia observed inthe NSDTR. While NSDTRs can be variably affected, the use of severelyaffected dogs enabled identification of the locus through GWAS.Haplotype sharing with chondrodystrophoid breeds and genome-wideassociation analysis for type I IVDD identified the same region onCFA12. Evaluation of mismapped mate pairs allowed the identification ofa novel FGF4 retrogene, which leads to an about 20 fold increase inexpression of FGF4 in neonatal intervertebral disc. Due to the embryonicexpression pattern of FGF4, it is probable that these expression changesare also impacting endochondral ossification. This is the second FGF4retrogene identified in dogs that affects limb length. While the FGF4retrogene on CFA18 impacts limb length, the FGF4 retrogene on CFA12explains the chondrodystrophoid phenotype, which includes limb lengthand IVDD (significant odds ratio >50).

Fibroblast Growth Factor 4 (FGF4) is a growth factor gene expressed inspecific tissues and at specific times throughout embryonic developmentin the mouse (39). FGF4 is highly expressed in the apical ectodermalridge of the developing limb bud, as well as somites and the notochordthat will form the vertebral column and IVDs (39-41). FGF signaling isrequired for appropriate embryonic axial growth and segmentation, andFGF4/FGF8 murine hypomorphs are characterized by altered vertebralmorphology and smaller limb buds (42, 43). Additionally, FGF8 hypomorphsare observed to have either hypoplastic or non-existent external earstructures (44). In mice, creation of a gain of function FGF4 copy toreplace an inactive FGF8 gene was able rescue limb development; however,it also caused abnormal tissue deposition and postaxial polydactyly,highlighting that levels of FGF throughout embryonic development must beproperly controlled for normal limb formation (32). While the specificembryonic expression pattern of FGF4 in dogs with 4-6 copies of the geneis unknown, we hypothesize that the insertion site milieu on CFA12versus CFA18 is contributing to differences in expression between theretrogenes, leading to the differences in phenotype.

A survey of retrogenes in the canine reference genome reported about 70functional retrogenes in the dog; however, only the previous CFA18 FGF4retrogene insertion has been reported to be associated with a diseasecausing phenotype (26, 45). Similarly in humans, the formation ofprocessed pseudogenes in general, as well as those that retain theirintended function and cause disease, is rare (46-51).

Both copies of the canine FGF4 retrogenes have signatures of havingarisen from RNA retrotransposed by LINE-1 integrase and reversetranscriptase, including flanking TSDs and polyA tracts (class 1templated sequence insertion polymorphism) (52). The CFA18 FGF4retrogene insertion was predicted to be expressed due to insertion nearsequence with promoter properties (26). While the CFA12 FGF4 insertionis placed near a potential TATA box and RNA Pol II promoter, it is morelikely that the CpG island included in the retrogene is drivingexpression (53-55). This hypothesis is supported by the finding that amajority of retrogene expression is actually due to genomic context andcontribution of CpG islands, not through the use of nearby promoters(56). To our knowledge, this is the first documentation of a secondretrogene insertion of the same parental gene resulting in a diseasephenotype in a mammalian species. Due to the lack of resources availableto identify these types of mutations, it is likely that there are otherphenotype inducing retrocopies present in the canine genome that haveyet to be discovered.

Chondrodystrophy associated mutation events occurred a very long timeago, as there are descriptions of short-legged dogs dating back over4000 years (57). In addition, both mutations occur concurrently in veryunrelated dog breeds from diverse breed groupings and geographicallocations. The fact that FGF4 has been retrotransposed twice in dogs inthe last 3-4 thousand years makes it likely that this has happened atother times. The large CpG island in the 5′ end of the endogenous FGF4gene may enable phenotypic consequences more readily than for otherretrogenes. Once the FGF4 retrogene appeared and produced an obviousphenotype, strong selection was likely applied to retain it, aided bythe semi-dominant nature of the mutation.

The NSDTR is the smallest of the retriever dog breeds, and based on theassociation of the CFA12 FGF4 insertion with height, we hypothesize thatthe heterozygous phenotype is aesthetically desirable and that selectionis maintaining the insertion at a relatively high allele frequency.Investigation of the CFA12 FGF4 insertion in additional breeds alsoshowed high allele frequency in multiple small and medium sized dogbreeds. In breeds also containing the CFA18 FGF4 insertion, there is aneven more dramatic decrease in height (e.g. Basset Hound, Cardigan WelshCorgi, Dachshund, etc.), supporting that both FGF4 retrogenes affectlong bone length.

In addition to segregating with height, the CFA12 FGF4 insertion alsosegregates with Hansen's type I IVDD susceptibility. Of the IVDD casesgenotyped for the CFA12 FGF4 insertion, all were homozygous mutant orheterozygous, except for 1, suggesting that one additional copy of FGF4on CFA12 is sufficient to cause type I IVDD. The single discordant casewas a Rottweiler, a breed that does not fit the chondrodystrophicphenotype. It is possible that there is another cause of IVDD innon-chondrodystrophoid dog breeds occurring without endochondralossification defects (9). IVDD-affected NSDTRs were also all eitherhomozygous or heterozygous for the CFA12 FGF4 insertion. Given that theCFA18 FGF4 insertion is not found in the NSDTR and was inconsistentlypresent in the IVDD cases tested, this further supports that theidentified insertion on CFA12 is causing both short limbs and Hansen'stype I IVDD in both the NSDTR and across dog breeds.

The breeds with a higher frequency of the CFA12 FGF4 insertion are thesame breeds identified in the last 50 years as being predisposed toIVDD. Presence of the CFA18 FGF4 insertion is common in many breeds withIVDD, and it is possible that it may contribute to the disease; however,previous mapping within Dachshunds, which are reported “fixed” for theCFA18 FGF4 insertion, actually show segregation of the associatedhaplotype on chromosome 12 with IVDD, supporting that the CFA12 FGF4insertion is the critical factor determining disease status (26, 35). Ofparticular interest is the lack of reports of IVDD cases in breeds suchas the Cairn Terrier and West Highland White Terrier, both of which havethe CFA18 FGF4 insertion, but not the CFA12 FGF4 insertion. Similarly,the high incidence of IVDD in breeds such as the American CockerSpaniel, Beagle, and French Bulldog that do not have the CFA18 FGF4insertion but a high frequency of the CFA12 FGF4 insertion supports thatFGF4 specifically from CFA12 is contributing to the IVDD phenotype.

The segregation of the CFA12 FGF4 insertion within dog breeds presentsan opportunity for improvement of animal health, as implementation ofgenetic testing over time could lead to the elimination of type I IVDD.Based on the ever-growing popularity of some breeds, the number ofanimals with this intervertebral disc disease mutation across the globeis in the millions. Myelopathy secondary to IVD herniation is the mostcommonly presenting neurological disorder of the spinal cord in dogs(58). The overall heath and financial consequences across the spectrumof presentations in companion dogs is immense. Prevention of diseasethrough breeding and eradication has the potential for far-reachingbenefits beyond those achievable through advances in surgical or medicaltherapy.

Additionally, the dog may serve as a valuable human-animal model forIVDD. Administration of a tyrosine kinase inhibitor in a mouse modelwith a gain of function mutation in FGFR3 has been shown to overcomegrowth defects associated with altered FGF signaling (33). Based on thephenotype and molecular etiology of chondrodystrophy and IVDD in dogs,it has the potential to serve as a bridge between mouse and humanstudies evaluating the efficacy of targeted pharmacological treatment ofFGF based genetic disorders.

Given the high mortality rate of IVDD and the high cost of surgery,identification of this susceptibility locus could provide a valuabletool for owners, breeders, and veterinarians for mitigating risk ofintervertebral disc herniation and resulting myelopathy (9). This couldbe especially useful in breeds that have both the CFA12 and CFA18 FGF4retrogene, as they could breed away from the CFA12 FGF4 retrogene, whilestill maintaining the aesthetically desirable shortness in staturecontributed by the CFA18 FGF4 retrogene. In breeds with only the CFA12FGF4 retrogene, breeders will ultimately decide if prevention ofHansen's type I IVDD outweighs any potential loss of shortness (or gainin height).

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTINGSequence ID No: 1 - 5′ Breakpoint of FGF4 insertion on CFA12at 33,710,178. Bolded and underlined text at nucleotideresidues 1002-2001 shows insertion sequence.TTCCTCAGACTCAGTTTCCCATTTGAAAGCAAGAAACTTTAGTTGGGCACTAGCCATTTCAGTTGGATTCCTTACTATGTCTTACTTCTCCCAAGAAGTTATTCCTTCCTATCTATCCATTGTATTAAGAAAGGCAGATTAGTTTTCCCATAGCCATCTGTTTATTTGGCCTATTGTCTTATGATTTCTCAAAACCAACACAGGTATTCCCAGGGTCATCTGTGACATCTGCCAAGCTGCCTGGGGAGCAGAGTCATATCTAGACATAACATGTGAAGGAGGTCATGTTTTGGTTGGCGGGGAGCTTTTTGGTTCTTTTTCTACCCAGAAGTCTCTTTTGTCTCCTTAGATGTCTCTTCTGTATTTATAGCTAGTCTTGGAGCATTGCTTTTTCCTACCTGTAAACTAAAGCGACAGTTCATAAAACTTCATATGTCTTTTTTTCTCTTACTCATTTTGTTTTGTTTTACTTTTCCCAATTATAAATTACATGACATTTAGAGAAAATTTGGAAAATATAAAGTTAAAAAGTTATAGACAGTACAACTCCCACACATAATCATGGCTAACATGGTGTATTTTTTATTCCTTCTTGGCTTGTATTTAGGTGTAATTTAAGAAAACTCTTCAAAATCCAAAGGGATAAGTTTAAGCAATTAAACAAATGGAACTGGTGAATAACAGCTGGCATGGTCAGTTATTTCTGTCTGAGGATCAGGGAGGGTTTGATGGAAGAGTTAATATTTCTGTTAGGTTTGAGAGTATAAATAGAATTTTGAAGAAGAGTGAATAAGGAAAAAAAATCCTATGATAAAAGAAAAACGTGGAAGCATTAAAGTATACATATATATGAATAATTAATGGCTGACTGTGGTCCTGAAGACTCTATTAAAATTTTGTGCTTTAGTTTCCTTGTTTATGCATTGGGGAGAGTCATATTTGTCTTTTTATCTTTCTAGAAAATGGAATATGTTAAGATAATTCCTATTCAAGTGCTTTGA GGCGGAGGGAGGCGCGCACCGCTCCGGAGGGTCCCAGCCCGGCCGCGCGTCCCGCCCGCCGCCCGCCGCCCGCCGCTCCATGCAGCCCGGGTAGCCCCGGCGCCCGGGGGCCCCGCGCCTCGCCTCCCGCTCCGCCTGCGGCCGCGCGCTCCGCACCGAGTCCCGGCCGTGCGCTCCCGCGGGCCGCCACAGGCGCAGCTCGGCCCCGCGGCTTCCCGGGCGCACGGCCCGAGGGCGGGGATGGCGGGGCCCGGGGCGGCCGCGGCGGCGCTGCTCCCGGCGGTCCTGCTGGCGGTGCTGGCGCCCTGGGCCGGCCGCGGGGGCGCCGCCGCTCCCACCGCCCCCAACGGCACGCTGGGCGCCGAGCTGGAGCGCCGCTGGGAGAGCCTGGTGGTGCGCTCGCTGGCGCGCCTGCCGGTGGCCGCGCAGCCCAAGGAGGCGGCCGTCCAGAGCGGCGCCGGCGACTACCTCCTGGGCATCAAGCGGCTGCGGCGGCTCTACTGCAACGTGGGCATCGGCTTCCACCTCCAGGTGCTCCCCGACGGCCGCATCGGCGGCGTGCACGCGGACACGAGCGACAGCCTGCTGGAGCTCTCGCCGGTGGAGCGGGGCGTGGTGAGCATCTTCGGCGTGGCCAGCCGGTTCTTCGTGGCCATGAGCAGCAAGGGCAAGCTGTACGGCTCGCCCTTCTTCACCGAGGAGTGCAAGTTCAAAGAGATCCTGCTCCCCAACAACTACAATGCCTACGAGTGCTGCAGGTACCCGGGCATGTTCATTGCCCTGAGCAAGAACGGGAAGACCAAGAAAGGGAGCCGAGTGTCCCCCACCATGAAGGTCACCCACTTCCTCCCCAGGCTGTGACTCCAGGCATCCTGCCTCAGTTTCCCAATGCTCCGGAGACTTTCTCCAGATGGACAATTTAATGCCAGAGTAGGTGTAAGATATTTAAATTAATTATTTAAATGTGTATATATCGCCACCAAATTATTTATGGTTCTGTSequence ID No: 2 - 3′Breakpoint of FGF4 insertion on CFA12 at33,710,178. Bolded and underlined text at nucleotideresidues 1-1000 shows insertion sequence.AGATGGAAGAGGCAGGGTCGGTGATGTTTAAAAAAAGTCCTGAGGTGATGGCAAACATTTAATTTTAATGAATGACTTTTTAGAGTTTATACAAAATGACCTTAGCTCGCTACCAGAAATGCTCCGAATGTTTTGTCAAGACTTTAATGCTCTCCTAGGATGTTTCTGAACCACCTCCCAAATTAACTTTATGGGAGTCTACAGACAGCAAGACTGGAAAAGGCTGATTGGAGTTTGTGTCTTTCGCATTCCTTTTTAAAACTCTTTGTTCGAATGCAAATCATCTACTTAAAATACTGTCCTTAAACCAAGGCCTTGGAGGAGGAAGGAAGCCGCTCGTGAAGCCTGATGGCTGGACTGTACATCTCAACCGGCCGTCCCCGTCCGTGCGGTGAAATAAAAAATGTTTTCAATTTTAAATTGCGTCCTAGGCTCCAGGAGTCTTGAGCAGAGGGGATGCTCCCAGGTCTCGGTGCTGATGGGGGGGAGGGGGCGGGGCTGGAATGTGTGGACATTCGGTATTTCAAATACTCGCCTCCTAAGTCTTAGCTGCCTTGGGATGATGGCACGATGTCTCATCTCAGAGCCCAATCCGATTGTCAGGAACGAAGATGTCTTAAGTGCAGAATGTGGTGATCCTTGGCCACTTGCTAGTCAGCGAGCCTTGTGGGAAGCGTATAGAGATGTCATTGGACCTCTGCAATATCGCTAAGTGTTTTCTACTGTCGTGATGGGATCTAAGGTTTCTGTACTTTCCGCGGTTTGCAGGATCTGTCTGTAGTTTTATACAGGTGCTGAGCCCTATTGTGATGTATGTGCTGTGCACATTGACATATGCCGAATAAATGGAAACATTTGTCATGTATGAAAAGAACCCCATTGGACTTGATGTAAAGAACCGGGGAAGGTATTGAAAACGATTAAAACCTGCCTGGAAATGCCATGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTGCTTTGA ACTCTTCAAAGAAAAGACACCTCAAAATCTACAGCATAATTGAGGTATCAGATTTTTGCCACTTTTTACTATTATGATTTTATTCACTAGCATTTATTTTGTCATCTAGAATTTTATTCACAAGCAAAAGCTTTTAGGAAATCATACTTATTTTAGGTTAAATGAGAAAAAACATTAGCTTAATTTTTTCTTAGTTTTTTTAATGTTTCTGACTATGATATAATACCTCACAATAGCTTATAAAGTGAATTTTCAATTTCAGTCATTATTAATTTGGCTGTCTCAAAATCAGGCTATATGTTATTCAAGTACCACTCTTTAGTGTCGTGAAAAAAATCAGTACAGTGGGTTCAAAAACAAACATCTTAATTGCAGACACTTGCAAATAGTTCAGAGTATGTGTTAATATGACATCTGTAACATTTCAGATATTTCTTGTTCTCAGTTTTTAAAAAAGCAATCTAAAAAGCAACGGAACAAAGGGAAAATATTGACAAGTAATCTGCTTAAAAACCCATGTGAATACGTGGAAATGTCAGATCTTTACACACTGTAATCTTGAACTTCCCATGTTTTATATGCTAAACATTCTAGCCTCTTGTTAGTTACCAGGTCTTTTAAAATTGTATGGGATTACATTCATATTTAAGATTGTATTCTGATTTTGCATGGACTAAATAAAAATCTACTTATTTAGACTGACAGTAATTTCACTGTCATTCTGCAAGTTGGGTTTTGTAGTTTTAGTAATCAAAAGTAGGTTTACTTTTTTAAAAAACTGTAATTCAGGTGTGGTATAGAAAATAAACTCAAGTGGGTAAGAAACATCTCAGTTCTTATTGCCTTTAAATGAAAATTAAACTAACTGATTTCAACCTCATCAGACTTCCAGGAAACCCAATCTCAATCAGTGATGGAGTCCCTGATTCTCTTAGGATCATAGCATCTGGGAAGTTCACATGGTATTGGATAAGGAAAAGGGCCTCTGCTGGCTAGGCCASEQ ID NO: 11 - oligo for detecting 5′-end of insertTCTTTCTAGAAAATGGAATATGTTAAGATAATTCCTATTCAAGTGCTTTGA GGCGGAGGGAGGCGCGCACCGCTCCGCAGGGTCCCAGCCCGGCCGCGCGWherein 5′-end of insert is at nucleic acid residue 52, andthe insertion sequence is in bold and underlined. Theoligonucleotide of SEQ ID NO: 11 is at least 20 nucleic acidsin length, comprising at least 3 nucleic acid bases flankingeither 5′ or 3′ of the 5′-end of insert (nucleic acid residue number 52)SEQ ID NO: 12 - oligo for detecting 3′-end of insert AAAAAAGTGCTTTGAACTCTTCAAAGAAAAGACACCTCAAAATCTACAGCATAATTGAGGT ATCAWherein 3′-end of insert is at nucleic acid residue 15, andthe insertion sequence is in bold and underlined. Theoligonucleotide of SEQ ID NO: 12 is at least 20 nucleic acidsin length, comprising at least 3 nucleic acid bases flankingeither 5′ or 3′ of the 3′-end of insert (nucleic acid residue number 15)

What is claimed is:
 1. A reaction mixture comprising (i) a biologicalsample from a canine comprising a nucleic acid template, and (ii) one ormore oligonucleotide pairs configured to detect the presence or absenceof a retrogene insertion encoding canine fibroblast growth factor 4(FGF4) on canine chromosome
 12. 2. The reaction mixture of claim 1,wherein the retrogene comprises about 3.2 kilobases (kb).
 3. Thereaction mixture of any one of claims 1 to 2, wherein retrogeneinsertion is inserted at a target site duplication sequence located atchr12:33,710,168-33,710,178 (canFam3).
 4. The reaction mixture of anyone of claims 1 to 3, wherein the oligonucleotide pairs detect the5′-end and/or the 3′-end of the retrogene insertion.
 5. The reactionmixture of any one of claims 1 to 4, wherein the 5′-end of the retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) comprises anucleic acid sequence having at least 90% sequence identity to nucleicacid residues 1002-2001 SEQ ID NO:1.
 6. The reaction mixture of any oneof claims 1 to 5, wherein the one or more oligonucleotide pairs areconfigured to detect the 5′-end of the retrogene insertion located atnucleic acid residue 1002 of SEQ ID NO:1.
 7. The reaction mixture of anyone of claims 1 to 6, wherein an oligonucleotide in the one or moreoligonucleotide pairs hybridizes to a sequence segment within nucleicacid residues 1-1001 of SEQ ID NO:1.
 8. The reaction mixture of any oneof claims 1 to 6, wherein the 3′-end of the retrogene insertion encodingcanine fibroblast growth factor 4 (FGF4) comprises a nucleic acidsequence having at least 90% sequence identity to nucleic acid residues1-1000 SEQ ID NO:2.
 9. The reaction mixture of any one of claims 1 to 8,wherein the one or more oligonucleotide pairs are configured to detectthe 3′-end of the retrogene insertion located at nucleic acid residue1000 of SEQ ID NO:2.
 10. The reaction mixture of any one of claims 1 to9, wherein an oligonucleotide in the one or more oligonucleotide pairshybridizes to a sequence segment within nucleic acid residues 1001-2000of SEQ ID NO:2.
 11. The reaction mixture of any one of claims 1 to 10,wherein the one or more oligonucleotide pairs comprise one or moreforward primers selected from the group consisting of:ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2), GTGTTTGCATGGAGGAAGGT (SEQ ID NO:3),CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4), AGCCTGATGGCTGGACTGTA (SEQ ID NO:5)and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and one or more reverse primersselected from the group consisting of TGCTGTAGATTTTGAGGTGTCTT (SEQ IDNO:7), CCTGATTTTGAGACAGCCAAA (SEQ ID NO:8), TTGATGCCCAGGAGGTAGTC (SEQ IDNO:9) and TGAGTGGGTTAAGGGTTTCG (SEQ ID NO:10).
 12. The reaction mixtureof any one of claims 1 to 11, wherein the one or more oligonucleotidescomprise one or more forward primers selected from the group consistingof: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2) and GTCCGTGCGGTGAAATAAAA (SEQ IDNO:6) and reverse primer TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7).
 13. Thereaction mixture of any one of claims 1 to 12, wherein the nucleic acidtemplate comprises genomic DNA.
 14. The reaction mixture of any one ofclaims 1 to 13, wherein the reaction mixture further comprises apolymerase and dNTPs.
 15. A kit comprising one or more oligonucleotidepairs that specifically identify the presence or absence of a retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) on caninechromosome
 12. 16. The kit of claim 15, wherein the oligonucleotidepairs detect the 5′-end and/or the 3′-end of the retrogene insertion.17. The kit of any one of claims 15 to 16, wherein the 5′-end of theretrogene insertion encoding canine fibroblast growth factor 4 (FGF4)comprises a nucleic acid sequence having at least 90% sequence identityto nucleic acid residues 1002-2001 SEQ ID NO:1.
 18. The kit of any oneof claims 15 to 17, wherein the one or more oligonucleotide pairs areconfigured to detect the 5′-end of the retrogene insertion located atnucleic acid residue 1002 of SEQ ID NO:1.
 19. The kit of any one ofclaims 15 to 18, wherein an oligonucleotide in the one or moreoligonucleotide pairs hybridizes to a sequence segment within nucleicacid residues 1-1001 of SEQ ID NO:1.
 20. The kit of any one of claims 15to 19, wherein the 3′-end of the retrogene insertion encoding caninefibroblast growth factor 4 (FGF4) comprises a nucleic acid sequencehaving at least 90% sequence identity to nucleic acid residues 1-1000SEQ ID NO:2.
 21. The kit of any one of claims 15 to 20, wherein the oneor more oligonucleotide pairs are configured to detect the 3′-end of theretrogene insertion located at nucleic acid residue 1000 of SEQ ID NO:2.22. The kit of any one of claims 15 to 21, wherein an oligonucleotide inthe one or more oligonucleotide pairs hybridizes to a sequence segmentwithin nucleic acid residues 1001-2000 of SEQ ID NO:2.
 23. The kit ofany one of claims 15 to 22, wherein the one or more oligonucleotidepairs comprise one or more forward primers selected from the groupconsisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2), GTGTTTGCATGGAGGAAGGT(SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4), AGCCTGATGGCTGGACTGTA(SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and one or morereverse primers selected from the group consisting ofTGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7), CCTGATTTTGAGACAGCCAAA (SEQ IDNO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9) and TGAGTGGGTTAAGGGTTTCG (SEQID NO:10).
 24. A solid support attached to one or more oligonucleotidesthat specifically identify the presence or absence of a retrogeneinsertion encoding canine fibroblast growth factor 4 (FGF4) on caninechromosome
 12. 25. The solid support of claim 24, wherein the solidsupport is attached to an oligonucleotide that hybridizes to the 5′-endof the retrogene insertion located at nucleic acid residue 1002 of SEQID NO:1.
 26. The solid support of any one of claims 24 to 25, whereinthe solid support is attached to an oligonucleotide that hybridizes tothe 3′-end of the retrogene insertion located at nucleic acid residue1000 of SEQ ID NO:2.
 27. The solid support of any one of claims 24 to26, wherein the solid support is attached to an oligonucleotide havingat least about 80% sequence identity to SEQ ID NO:11 and/or SEQ IDNO:12.
 28. The solid support of any one of claims 24 to 27, wherein thesolid support is a microarray.
 29. The solid support of any one ofclaims 24 to 27, wherein the solid support is a mounted tissue sample.30. A kit comprising the solid support of any one of claims 24 to 28.31. A method for identifying a canine suffering from or at risk ofsuffering from skeletal dysplasia (SD) and/or intervertebral discdisease (IVDD), the method comprising: a) obtaining a biological samplecomprising a nucleic acid template from the canine; b) determining thepresence or absence of a retrogene insertion encoding canine fibroblastgrowth factor 4 (FGF4) on canine chromosome 12; and c) selecting acanine comprising the retrogene insertion identifies a canine sufferingfrom or at risk of suffering from skeletal dysplasia (SD) and/orintervertebral disc disease (IVDD) relative to canine that does not havethe retrogene insertion encoding canine fibroblast growth factor 4(FGF4) on canine chromosome
 12. 32. A method for identifying a caninewith reduced risk of suffering from skeletal dysplasia (SD) and/orintervertebral disc disease (IVDD), the method comprising: a) obtaininga biological sample comprising a nucleic acid template from the canine;b) determining the presence or absence of a retrogene insertion encodingcanine fibroblast growth factor 4 (FGF4) on canine chromosome 12; and c)selecting a canine that does not comprise the retrogene insertionidentifies a canine with reduced risk of suffering from skeletaldysplasia (SD) and/or intervertebral disc disease (IVDD) relative tocanine that has the retrogene insertion encoding canine fibroblastgrowth factor 4 (FGF4) on canine chromosome
 12. 33. The method of anyone of claims 31 to 32, wherein the retrogene comprises about 3.2kilobases (kb).
 34. The method of any one of claims 31 to 33, whereinretrogene insertion is inserted at a target site duplication sequencelocated at chr12:33,710,168-33,710,178 (canFam3).
 35. The method of anyone of claims 31 to 34, wherein the determining step employs one or morepolynucleotides configured to detect the 5′-end and/or the 3′-end of theretrogene insertion.
 36. The method of claim 35, wherein the 5′-end ofthe retrogene insertion encoding canine fibroblast growth factor 4(FGF4) comprises a nucleic acid sequence having at least 90% sequenceidentity to nucleic acid residues 1002-2001 SEQ ID NO:1.
 37. The methodof any one of claims 35 to 36, wherein the one or more polynucleotidesare configured to detect the 5′-end of the retrogene insertion locatedat nucleic acid residue 1002 of SEQ ID NO:1.
 38. The method of any oneof claims 35 to 37, wherein one polynucleotide hybridizes to a sequencesegment within nucleic acid residues 1-1001 of SEQ ID NO:1.
 39. Themethod of any one of claims 35 to 38, wherein the 3′-end of theretrogene insertion encoding canine fibroblast growth factor 4 (FGF4)comprises a nucleic acid sequence having at least 90% sequence identityto nucleic acid residues 1-1000 SEQ ID NO:2.
 40. The method of any oneof claims 35 to 39, wherein the one or more polynucleotides areconfigured to detect the 3′-end of the retrogene insertion located atnucleic acid residue 1000 of SEQ ID NO:2.
 41. The method of any one ofclaims 35 to 40, wherein one polynucleotide hybridizes to a sequencesegment within nucleic acid residues 1001-2000 of SEQ ID NO:2.
 42. Themethod of any one of claims 35 to 41, wherein the SD/IVDD genotype isdetected by an amplification reaction using polynucleotides thatidentify the presence or absence of the CFA 12 FGF4 retrogene insertion.43. The method of claim 42, wherein the amplification reaction isselected from the group consisting of polymerase chain reaction (PCR),strand displacement amplification (SDA), nucleic acid sequence basedamplification (NASBA), rolling circle amplification (RCA), T7 polymerasemediated amplification, T3 polymerase mediated amplification and SP6polymerase mediated amplification.
 44. The method of any one of claims31 to 43, wherein the 5′-end and/or the 3′-end of the retrogeneinsertion are detected.
 45. The method of any one of claims 31 to 44,wherein a portion of the retrogene insertion sequence is specificallyamplified using one or more forward primers selected from the groupconsisting of: ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2), GTGTTTGCATGGAGGAAGGT(SEQ ID NO:3), CTGAGCAAGAACGGGAAGAC (SEQ ID NO:4), AGCCTGATGGCTGGACTGTA(SEQ ID NO:5) and GTCCGTGCGGTGAAATAAAA (SEQ ID NO:6) and one or morereverse primers selected from the group consisting ofTGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7), CCTGATTTTGAGACAGCCAAA (SEQ IDNO:8), TTGATGCCCAGGAGGTAGTC (SEQ ID NO:9) and TGAGTGGGTTAAGGGTTTCG (SEQID NO:10).
 46. The method of any one of claims 31 to 45, wherein aportion of the retrogene insertion sequence is specifically amplifiedusing one or more forward primers selected from the group consisting of:ACAGCTGGCATGGTCAGTTA (SEQ ID NO:2) and GTCCGTGCGGTGAAATAAAA (SEQ IDNO:6) and reverse primer TGCTGTAGATTTTGAGGTGTCTT (SEQ ID NO:7).
 47. Themethod of any one of claims 31 to 46, wherein the SD/IVDD genotype isdetected by hybridization using polynucleotides which identify thepresence or absence of the CFA 12 FGF4 retrogene insertion.
 48. Themethod of any one of claims 31 to 47, wherein the SD/IVDD genotype isdetected by sequencing.
 49. The method of any one of claims 31 to 48,wherein the canine is a domesticated canine.
 50. The method of any oneof claims 31 to 49, wherein the canine is of a breed having apredisposition to chondrodystrophy.
 51. The method of any one of claims31 to 50, wherein the canine is a purebred or mix from a breed selectedfrom the group consisting of American Cocker Spaniel, Basset Hound,Beagle, Cardigan Welsh Corgi, Chesapeake Bay Retriever, Chihuahua, Cotonde Tulear, Dachshund, English Springer Spaniel, French Bulldog, JackRussell Terrier, Miniature Schnauzer, Nova Scotia Duck TollingRetriever, Pekingese, Pembroke Welsh Corgi, Poodle, Portuguese WaterDog, Scottish Terrier, Shih Tzu, and mixtures thereof.
 52. The method ofany one of claims 31 to 50, wherein the canine is a purebred or mix froma breed selected from the group consisting of American Cocker Spaniel,Basset Hound, Beagle, Corgi, Dachshund, French bulldog, Nova Scotia DuckTolling Retriever, and Pekingese.